Chromosomal markers and diagnostic tests for manic-depressive illness

ABSTRACT

Methods and compositions are provided for determining a genotype associated with increased susceptibility to manic-depressive illness. The genotype is determined using markers for a region of chromosome 18 exhibiting linkage disequilibrium with manic-depressive illness. The invention also provides for a novel myo-inositol monophosphatase protein encoded for on chromosome 18.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a Continuation-In-Part application(“CIP”) of U.S. Provisional Application Serial No. 60/029,278, filedOct. 28, 1996. The aforementioned application is explicitly incorporatedherein by reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods fordetermining the genotype associated with an increased or decreasedsusceptibility to manic-depressive illness. The invention also providesa means to determine an individual's increased or decreased risk ofdeveloping manic-depressive illness.

BACKGROUND OF THE INVENTION

[0003] Genome screening efforts by several groups, designed to identifyregions linked to bipolar disorder, have revealed evidence for potentialsusceptibility loci on chromosome 18. Berrettini (1994) Proc Natl AcadSci USA 91:5918-5921, reported evidence for a susceptibility locus inthe pericentromeric region of the chromosome. In a subsequent study onan independent pedigree series, Stine (1995) Am. J. Hum. Genet.57:1384-1394, found support for the prior hypothesis on 18p (Berrettiniet al., Proc Natl Acad. Sci. USA, 91:5918-5921, 1994). In the samestudy, Stine (1995) supra, presented evidence for a possible additionallinkage on 18q. Recently, Freimer (1996) Nature Genet. 12:436-441,proposed a predisposing locus close to the telomere of 18q in CostaRican kindreds. These reports suggest that the regions potentiallyimplicated in bipolar disorder encompass a very large portion ofchromosome 18.

[0004] In addition to bipolar disorder, more than 25 other diseases havebeen localized to chromosome 18, approximately 80% of which still awaitthe discovery of the underlying defective gene (Overhauser et al.Cytogenet Cell Gene, 71:106-117. 1995; Online Mendelian Inheritance inMan (OMIM) (TM). (Database on line, 1995; URL:http://www3.ncbi.nlm.nih.gov/omim/. cited Jan. 19, 1996)). Since thischromosome has a genetic length estimated to be 150 cM [CooperativeHuman Linkage Center (CHLC), Science 265:2049-2054, 1994], whichincludes about 4.5% of the total length of the genome, it is expected toencode several thousand genes. Approximately 40 genes have been mappedto this chromosome [Overhauser et al., 1995; Genome Database (GDB), URL:http://gdbwww.gdb.org/gdb/browser/docs/topq.html>[database online].(1990-). Updated daily (cited Jan. 19, 1996]; OMIM, [Database on line,1995; cited Jan. 19, 1996]. Between 1993 and 1995, only 14 genes havebeen added to the list of chromosome 18 genes (Geurts van Kessel et al.,Cytogenet Cell Gene, 65:141-165, 1994; Overhauser et al., Cytogenet CellGene, 71:106-117, 1995). Therefore, a dense transcriptional map, whichwould be valuable in positional cloning of susceptibility genes, remainsto be developed for chromosome 18.

SUMMARY OF THE INVENTION

[0005] In one aspect the present invention is directed to a method fordetermining a genotype associated with increased susceptibility tomanic-depressive illness. The method comprises determining the genotypeof an affected individual with at least one polymorphic marker localizedwithin the chromosomal region defined by and including markers D18S843and D18S869, and determining therefrom the genotype associated withincreased susceptibility to manic-depressive disorder.

[0006] In preferred embodiments the polymorphic marker is amplified byprimers which selectively hybridize, under stringent conditions, to thesame nucleic acid sequences as primers of SEQ ID NO:1 and SEQ ID NO:2(see Table 1, below, forward and reverse primers to amplify Clone 22).Typically the polymorphic marker is amplified by the polymerase chainreaction.

[0007] In other embodiments the method of further comprises determiningthe genotype of a tested individual wherein the genotype is determinedwith at least one polymorphic marker localized within the chromosomalregion defined by and including markers D18S843 and D18S869. Thegenotype of the tested individual is compared to the genotype associatedwith increased susceptibility to manic-depressive illness and theincreased or decreased risk of the tested individual developingmanic-depressive illness is TABLE 1 PCR PRIMER SEQUENCES Name PrimersSEQ ID NO: Name Primers SEQ ID NO: Clone 22 F-TACAAAAGAGGACAAAGCAC 30D18S73 F-TGCCACTGCAACAATGC 31 R-GGTGCCTGTATATAAGTTGA 32R-CCCAGCAATCAACCTTTAAG 33 Clone 24 F-CTACAGAATAGAATACATGGCG 34 D18S869F-TGTTTATTTGTTTGACTCAATGG 35 R-GAGCTCTGAACTGTATTCAGA 36R-GAGTGAATGCTGTACAAACAGC 37 Clone 29 F-TCTCAGCTTACTCAACCT 38 D18S996F-GATGGAAAGCCATTTTATTTTTC 39 R-GATGAGGTGGAACAATCAC 40R-TCGTACTATGAAATTTTTAAGCCTT 41 GNAL F-GGTCTGTACAGTGTAATAAACC 42 FB14A10F-CCTTCCCCTCTATTCTCAAA 43 R-CTACTGCAAAATGTGTCCTGTC 44R-GAGCGAGACTGTCTCAAAAA 45 Clone 37 F-CACATTAGCCAGTCTGATAAAG 46 GC32001F-GAGTTGTGGGGGGGAATAGT 47 R-AAGTTACACACAGTAGCTGA 48R-ATACGGAGGTTGAACTAGGAAGG 49 AFMa058yg5 F-TAGATGCTATATTAGGCTGGGTCTC 50GP4B15 F-CGGTTCTGGATTTATCAGTA 51 R-GAACTTACAGCACTGGCTCTCC 52R-AGGGTTGCAATGAGCTGAG 53 AFMa152wg9 F-AAGAACAAAAGGTCACCTGTCA 54 IB-1114F-GCCACACACAAATTTTTCTC 55 R-TGTCTCACCTCTGCTCACTCAT 56R-ACAGGGTGTAAGAGGAGAGG 57 CHLC.GGA16G02 F-ATGGAAGGAXAAACAGAGGG 58NIB-1802 F-CTGATCACATTTCATACAGC 59 R-GAACTCTTCAAGAGGGGAGC 60R-TGTATGTGGGCTTAACTGTT 61 D18S1114 F-ATCAGTATAATGATGGATGAATCAC 62SGC-31363 F-CTACTGGGAGGTAGGTAATCTCAG 63 R-TGAGGCAAGAGGGTCAC 64R-GCAAAACCAACCACATCAAA 65 D18S1116 F-TCTGCCACTTTTTATGGG 66 SGC34207F-GATCCTGTTCTTTCAGCAGG 67 R-CAATGTTTTAACTTCTAGGACAAAT 68R-TTTAACCAGCTGGAGTGAAGG 69 D18S1150 F-GGCACAGGAAACGTGAAT 70 WI-11680F-ACAGATACTTTTCCACGCAACA 71 R-CACAAGGATGCCAGCC 72R-AAAAAGATGTACGGTCTGGCC 73 D18S1153 F-ATGGAGGCTCTGAGACCCTT 74 WI-13171F-TTTTATTTGGACAAGAGAACTTGTG 75 R-CTTGCCTGATGCCTGAAAT 76R-ATGATCAGCTCTGAGGTGCA 77 D18S1158 F-GCATCTATGCAGTGCCAAAT 78 WI-18080F-TGGCATAAAGTTTGCAAATATCA 79 R-TCATTAGCAACAAGGATCTCC 80R-ATACACCAAAGGAGAAGGATTAACA 81 D18S1228 F-AGACAGTTGAAAAGGACACAAATG 82D18S1066 F-TGCTGTTGCCTCTCAGCATCTC 83 R-TGGTGATGGGACTTTTCAAA 84R-CACCTTTCAAGTGCTTGGCAGTC 85 D18S378 F-AGCCTGGGTGACAGAGCAA 86 D18S1215F-GTTTGCTGCATCTCCCAATT 87 R-ACAGGGAAAGCTGGGGGAT 88R-GTGCCCACATTGTTGTGAAG 89 D18S40 F-CAAGATAGATGCATTTTCCAGT 90 D18S1299F-TTTAAGCCTCAAGGGACCCT 91 R-CATCCAAAGGGTGAATGTGT 92R-AGATTGAGGACCAGGTGGTG 93 D18S464 F-GCCAGACTTTGTGCCATTTC 94 D18S1226F-CTCTTAAGTTGAGTGAAGTGGAAGC 95 R-TTTCCTGAATCTCTTGTGGTTTG 96R-CGCAAAAGTCAGGAAAGAGG 97 D18S482 F-ATGAGTGAATGCCAACTTCG 98 SHGC-32282F-TTACGCATTTTGTATCAGACTTACA 99 R-CCTGGCTGACAGAGTGAGT 100R-GGTGGAGTATCAGAAGTGATTTTAG 101 D18S53 F-GGTCACCTACAACTTTGGATG 102D18S1315 F-TGGACTTCTACCCCCATCTG 103 R-TGCATGTAAATATCAGAGTCTGTT 104R-TTTGAAACCTGGACACTTTGG 105 D18S71 F-ACCCGCTCAAAAGCCT 106 D18S843F-GTCCTCATCTGTAAAACGGG 107 R-TTAATGGATTATCAAGAGTGGTTCT 108R-CCACTAACTAGTTTGTGACTTTGG 109

[0008] determined therefrom. Generally, the polymorphic marker of thetested individual is amplified by primers which selectively hybridize,under stringent conditions, to the same nucleic acid sequences asprimers of SEQ ID NO:11 and SEQ ID NO:2.

[0009] In another aspect, the present invention is directed to a nucleicacid composition comprising oligonucleotide primers which selectivelyhybridize, under stringent conditions, to the same nucleic acid sequenceas primers of SEQ ID NO:1 and SEQ ID NO:2. In an additional aspect thepresent invention is directed to a nucleic acid of less than 10 kB andcomprising a polymorphic marker amplified by oligonucleotide primers ofSEQ ID NO:1 and SEQ ID NO:2.

[0010] In yet another aspect, the present invention is directed to amethod for determining an increased susceptibility to manic-depressiveillness in an individual, comprising determining the genotype of theindividual with oligonucleotide primers. The oligonucleotide primersamplify a polymorphic site as primers of SEQ ID NO:1 and SEQ ID NO:2.This polymorphic marker can be found in at least two forms, designatedas “allele 1” of clone 22 (SEQ ID NO:14) or “allele 2” of clone 22 (SEQID NO:15). The presence of allele 2 of the polymorphic marker indicatesan increased susceptibility to manic-depressive illness.

[0011] The invention further provides for a isolated nucleic acidencoding an IMP.18p myo-inositol monophosphatase, the protein defined ashaving a calculated molecular weight of between about 22 to 34 ka, andwhere the protein's activity includes hydrolysis of myo-inositol1-phosphate to generate inositol and inorganic phosphate; and where theprotein specifically binds to an antibody raised against an IMP.18pmyo-inositol monophosphatase protein, or immunogenic fragment thereof,consisting of SEQ ID NO:17; or, having at least 60% amino acid sequenceidentity to an IMP.18p myo-inositol monophosphatase protein consistingof SEQ ID NO:17, as measured using a sequence comparison algorithm. Inone embodiment, the nucleic acid encodes a IMP.18p myo-inositolmonophosphatase having a calculated molecular weight of about 28 to 29kDa. In other embodiments, the isolated nucleic acid; encodes a proteinwhich has at least 80% amino acid sequence identity to the IMP.18pmyo-inositol monophosphatase protein of SEQ ID NO:17. as measured usinga sequence comparison algorithm; encodes a protein having the sequenceset forth in SEQ ID NO:17; specifically hybridizes to SEQ ID NO:16 understringent conditions; or, encodes an IMP.18p myo-inositolmonophosphatase protein which specifically binds to an antibody directedagainst a protein having a sequence as set forth in SEQ ID NO:17.

[0012] In further embodiments, the invention also provides for apolynucleotide or fragment thereof comprising a purified antisensenucleotide capable of hybridizing to and having a nucleic acid sequencecomplementary to at least a portion of an IMP.18p myo-inositolmonophosphatase polynucleotide. The invention also provides for anexpression vector comprising a nucleic acid encoding an IMP.18pmyo-inositol monophosphatase or its antisense sequence. Furtherembodiments provide for a cell comprising an exogenous nucleic acidsequence encoding an IMP.18p myo-inositol monophosphatase protein.Another embodiment provides for an organism into which an exogenousnucleic acid sequence which specifically hybridizes under stringentconditions to SEQ ID NO:16 or which comprises a nucleic acid encoding anIMP.18p myo-inositol monophosphatase or fragment thereof, has beenintroduced, and the organism expresses the exogenous nucleic acid as anIMP.18p myo-inositol monophosphatase protein, or fragment thereof. Inone embodiment, the organism's exogenous nucleic acid sequence istranslated into an IMP.18p myo-inositol monophosphatase protein which isexpressed externally from the organism.

[0013] The invention also provides for an isolated IMP.18p myo-inositolmonophosphatase protein having a calculated molecular weight of about 22to 34 kDa; where the protein's activity includes hydrolysis ofmyo-inositol 1-phosphate to generate inositol and inorganic phosphate;and specifically binds to an antibody raised against a myo-inositolmonophosphatase protein, or immunogenic fragment thereof, consisting ofSEQ ID NO:17, or has at least 60% amino acid sequence identity to amyo-inositol monophosphatase protein consisting of SEQ ID NO:17, asmeasured using a sequence comparison algorithm. In one embodiment, theisolated IMP.18p myo-inositol monophosphatase protein can also be foundin humans. In further embodiments, the isolated IMP.18p myo-inositolmonophosphatase protein has a calculated molecular weight of about 28 to29 kDa; or, has a sequence as set forth in SEQ ID NO:17.

[0014] The invention further provides for an isolated antibody which isspecifically immunoreactive under immunologically reactive conditions toan IMP.18p myo-inositol monophosphatase protein having the sequence asset forth in SEQ ID NO:17. In another embodiment, the isolated antibodyis specifically immunoreactive under immunologically reactive conditionsto an IMP.18p myo-inositol monophosphatase protein encoded by a IMP.18pmyo-inositol monophosphatase nucleic acid of the invention.

[0015] Also provided for in the invention is a pharmaceuticalcomposition comprising an acceptable carrier and an IMP.18p myo-inositolmonophosphatase protein; an anti-IMP.18p myo-inositol monophosphataseantibody or binding fragment thereof; or a polynucleotide encoding anIMP.18p myo-inositol monophosphatase protein.

[0016] The invention also provides for a method for quantifying theamount of a myo-inositol monophosphatase in a mammal, comprising:obtaining a cell or tissue sample from the mammal; and, determining theamount of an IMP.18p myo-inositol monophosphatase gene product in thecell or tissue.

[0017] Another embodiment provides for a method for detecting thepresence of a polynucleotide sequence encoding at least a portion of anIMP.18p myo-inositol monophosphatase in a biological sample, comprisingthe steps of providing a biological sample suspected of containing aIMP.18p myo-inositol monophosphatase-encoding nucleic acid and a probecapable of hybridizing to at least a portion of an IMP.18p myo-inositolmonophosphatase nucleotide sequence, or a fragment thereof, from abiological sample; then combining the nucleic acid-containing biologicalsample with the probe under conditions such that a hybridization complexis formed between the nucleic acid and the probe; and detecting thehybridization complex. In one embodiment the nucleic acid in thebiological sample is ribonucleic acid. In another embodiment, thedetected hybridization complex correlates with expression of an IMP.18pmyo-inositol monophosphatase in the biological sample.

[0018] The invention also provides for a method of determining whether atest compound is a modulator of an IMP.18p myo-inositol monophosphataseactivity, the method comprising the steps of: providing a compositioncomprising an IMP.18p myo-inositol monophosphatase protein; contactingthe monophosphatase with the test compound; and measuring the activityof the monophosphatase, wherein a change in monophosphatase activity inthe presence of the test compound is an indicator of whether the testcompound modulates monophosphatase activity. In one embodiment, thecomposition comprises monophosphatase is encoded a an IMP.18pmyo-inositol monophosphatase polypeptide of the invention. In furtherembodiments, the composition comprises a cell or an organism.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 shows the assignment of brain transcripts to chromosome 18cytogenetic bins. cDNA selection yielded a total of 48 brain-expressedtranscripts (numbered 1 to 48) that mapped specifically to the indicatedregions of chromosome 18. Redundant transcripts are in parenthesis nextto the first member of each redundant group. The somatic cell hybridsthat subdivide the chromosome into cytogenetic bins (represented by A toS, from pter to qter, right hand side) and the names of the cell lines(bottom) are indicated.

[0020]FIG. 2 shows the results of a high resolution mapping oftranscripts versus chromosome 18 reference STS by the use of radiationhybrids. A schematic representation of the position of the uniquetranscripts with respect to linked STSs. Transcripts and genes that aremembers of a radiation hybrid linkage group are enclosed in dashedboxes. The approximate locations within the cytogenetic bins are alsoindicated.

[0021]FIG. 3 shows the results of radiation hybrid mapping of the18p11.2 region. Distances are shown in centirays (cR). Vertical linesrepresent probable locations of the indicated markers. Thickenedvertical lines indicate the most probable location of the indicatedmarkers.

[0022]FIG. 4 shows that the amplified, radiolabeled probe from cDNAclone ID #39740 was found to detect a major band of approximately 1.5 kbin multiple tissues through Northern hybridization. FIGS. 4A, 4B, and 4Cshow hybridization in; fetal brain, lung, liver and kidney; adult heart,brain, lung, liver, skeletal muscle, kidney, and pancreas; and, adultbrain amygdala, caudate nucleus, corpus callosum, hippocampus,hypothalamus, substantia nigra, subthalamic nucleus, and thalamus.Control hybridizations with a GAPDH probe are also shown.

[0023]FIG. 5B shows the complete 1447 base pair full-length cDNAnucleotide sequence (SEQ ID NO:16) and the corresponding predicted aminoacid sequence (SEQ ID NO:17) of the novel IMP.18p of the invention. FIG.5A shows a schematic representation of this newly discovered messagealigned with clone #39740 (IMAGE Consortium).

[0024]FIG. 6 shows the alignment of the deduced amino acid sequence ofIMP.18p with other IMPs and protein motifs characteristic of themyo-inositol monophosphatase protein family.

[0025]FIG. 7 shows the mapping position of the gene encoding the IMP.18pmyo-inositol monophosphatase is within the bipolar susceptibility regionat 18p11.2 of chromosome 18.

[0026]FIG. 8 shows the promoter sequence for IMP.18p.

DETAILED DESCRIPTION OF THE INVENTION

[0027] In the present invention, a region of chromosome 18 has beenidentified that is tightly linked to a locus associated withsusceptibility to manic-depressive illness, including affectivedisorders. Linkage disequilibrium between a particular form of a markerin the population and the presence of the manic-depressive illnessprovides a means to determine the increased susceptibility of anindividual to manic-depressive illness. Accordingly, the methods andcompositions of the present invention provide a means to alertclinicians to a genetic predisposition towards developingmanic-depressive illness. The methods of the invention are useful ingenetic counseling of individuals from families affected withmanic-depressive illness, and aid in the differential diagnosis ofmanic-depressive illness from other psychiatric pathologies.

[0028] A susceptibility region for bipolar disorder has been found onthe pericentromeric portion of chromosome 18 (Berrettini (1994) Proc.Natl. Acad. Sci. USA 91:5918-5924). The invention provides the noveldiscovery that genes and markers corresponding to bipolar disease map tothe region of chromosome 18 designated region 18p11.2. This finding ledto the discovery of a novel gene encoded in 18p11.2 whose chromosomallocation is linked with bipolar disorder, as described in Example 13.

[0029] This novel, full-length cDNA, designated IMP.18p (alternativelydesignated IMPA2), was isolated and sequenced (SEQ ID NO:16, see FIG.5B), as described in Example 13. Its predicted polypeptide translationproduct is 288 amino acids (SEQ ID NO:17, see FIG. 5B). The deducedamino acid sequence revealed approximately 54% sequence identity with ahuman brain myo-inositol monophosphatase (IMP), as described byMcAllister, (1992) Biochem J. 284:749-754, GenBank, Accession #P29218(also designated IMPA1). The IMP.18p sequence also included motifscharacteristic of other IMP proteins (as described in detail below).Thus, the IMP.18p of the invention is a novel myo-inositolmonophosphatase (IMP) protein.

[0030] The invention also provides for novel anti-IMP.18p reagents inthe form of anti-IMP.18p antibodies and IMP.18p-encoding nucleic acidsto identify polymorphic variants of IMP.18p within the scope of theclaimed invention. Use these novel reagents in various antibody-basedand nucleic acid-based assays to clearly describe the identification andisolation of such polymorphic variants are described below.

[0031] To provide a more precise location of this gene, mapping with apanel of radiation hybrids (RH) was conducted. Multipoint RH analysisplaced the gene between GNAL and D18S71 within the 18p11.2 region (seeFIG. 3). Thus, IMP.18p is a gene localized within the chromosomal regiondefined by and including markers D18S843 and D18S869. Because of thephysical position of IMP.18p coding sequence on chromosome 18 and itspotential function, IMP.18p is an important gene for the treatment anddiagnosis of manic depressive illnesses, including bipolar disorder.

[0032] Lithium is the most commonly prescribed medication and effectivetreatment for manic depression/bipolar disorder. Its therapeutic actionis in part mediated through the inhibition of IMP, an enzyme which has acrucial role in the phosphatidylinositol signaling pathway (reviewed inAtack (1996) “Inositol monophosphatase, the putative therapeutic targetfor lithium.” Brain Res. Rev. 22:183-190; see also Ragan (1988) BiochemJ. 249:143-149). IMP is a homodimer, with each subunit organized in analpha beta alpha beta alpha arrangement of alpha-helices andbeta-sheets. This type of structure seems crucial to a two-metalcatalyzed mechanism. Lithium appears to inhibit the IMP enzyme followingsubstrate hydrolysis by occupying the second metal binding site before aphosphate group can dissociate from its interaction with the first metalsite.

[0033] As IMP is a molecular target for the therapeutic effects oflithium, inhibitors of IMP can be lithium-mimetics. Thus, the novelIMP.18p of the invention, which is distantly related to inositolmonophosphatase enzymes, can be used to not only to identify inhibitorsspecific for IMP.18p, but also as a novel means to identify and isolatenew inhibitors of IMPs as alternatives to lithium.

[0034] In disease states associated with increased levels of IMPactivity, such as bipolar disease, the enzymatic activity and levels ofIMP.18p is altered in specific brain areas. Thus, the IMP.18p nucleicacid sequence of the invention provides for novel means to measurelevels of IMP and diagnose the corresponding disease state.

[0035] Because of the location and function of IMP.18p, it qualifies asa novel target for diagnosis, therapeutics and molecular scanning, i.e.,identification of mutations, polymorphisms and further members of thisnew myo-inositol monophosphatase enzyme family.

[0036] Definitions

[0037] Units, prefixes, and symbols can be denoted in their SI acceptedform. Numeric ranges are inclusive of the numbers defining the range.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation. The headings provided herein are notlimitations of the various aspects or embodiments of the invention whichcan be had by reference to the specification as a whole. Accordingly,the terms defined immediately below are more fully defined by referenceto the specification in its entirety.

[0038] As used herein, “manic-depressive illness” and bipolar disorder,including bipolar I (BPI) and bipolar II (BPII), refer to the samephenotype and can be used interchangeably. Manic depressive disorderincludes reference to schizoaffective disorder, or recurrent MajorDepressive Illness (i.e., recurrent unipolar illness). See, “ResearchDiagnostic Criteria,” Spitzer et al. Arch. Gen. Psychiat., 35:773-779(1978); Endicott, J. and Spitzer, L., Arch. Gen. Psychiat., 35:837-862(1978); and, Diagnostic and Statistical Manual of Mental DisordersIII-R, (1980), American Psychiatric Association, Washington D.C.,Spitzer and Williarns (ed.), each of which is incorporated herein byreference. An individual affected by manic-depressive illness is an“affected individual.”

[0039] As used herein, “marker” includes reference to a locus on achromosome that serves to identify a unique position on the chromosome.A “polymorphic marker” includes reference to a marker which can appearin multiple forms, i.e., these different forms sometimes referred to as“alleles” (alleles are defined as different variations of a gene ormarker). Different forms of the marker can be used to follow theirtransmission from parent to child and throughout generations (when theyare present in a homologous pair, allow transmission of each of thechromosomes in that pair to be followed).

[0040] A genotype may be defined by use of a single or a plurality ofmarkers.

[0041] As used herein, “chromosomal region” includes reference to alength of chromosome which may be measured by reference to the linearsegment of DNA which it comprises. The chromosomal region can be definedby reference to two unique DNA sequences, i.e. markers.

[0042] As used herein, “genotype associated with increasedsusceptibility to manic-depressive illness” includes reference to agenotype which has a higher probability of occurrence in amanic-depressive illness affected individual than in members of thegeneral United States population who are past the age of onset butunaffected by manic-depressive illness.

[0043] As used herein, “increased” means greater than that of the U.S.population average. Thus, an increased susceptibility tomanic-depressive illness includes reference to a greater risk ofdeveloping manic-depressive illness than the average risk for the U.S.population.

[0044] As used herein, “decreased” means less than that of the U.S.population average. Thus, a decreased susceptibility to manic-depressiveillness includes reference to a lesser risk of developingmanic-depressive illness than the average risk for the U.S. population.

[0045] As used herein, “determining” the “risk of the tested individualdeveloping familial manic-depressive illness” means ascertaining theprobability of the tested individual developing manic-depressive illnessafter the individual reaches the age of onset. The determination of riskmay be a quantitatively assessed or may be assessed qualitatively ashigher, lower, or equivalent to the average risk to the U.S. population.

[0046] As used herein, “tested individual” includes reference to a humanwhose genotype is being determined. The tested individual may be pre- orpost-partum.

[0047] As used herein, “localized within the chromosomal region definedby and including” with respect to particular markers includes referenceto a contiguous length of a chromosome delimited by and including thestated markers.

[0048] As used herein, “manic-depressive illness genotype” includesreference to a genotype determined with at least one polymorphic markerwithin the chromosomal region defined by markers linked to the locusassociated with susceptibility to manic-depressive illness. Preferably,the genotype is deter-mined using polymorphic markers within 5centimorgans of the polymorphic marker defined by SEQ ID NO:1 and SEQ IDNO:2. In a preferred embodiment, the chromosomal region is defined(flanked) by and includes chromosomal markers D18S843 and D18S869. In aparticularly preferred embodiment, the genotype is determined using themarker amplified by oligonucleotide primers of SEQ ID NO:1 and SEQ IDNO:2 (Table 1).

[0049] As used herein, “isolated.” “purified” or “biologically pure”refer to material which is substantially or essentially free fromcomponents which normally accompany or interact with it as found in itsnaturally occurring environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment. Purity and homogeneity are typically determined usinganalytical chemistry techniques, e.g., sequence analysis, gelelectrophoresis or high performance liquid chromatography (HPLC). Aprotein that is the predominant species present in a preparation issubstantially purified. In particular, an isolated IMP.18p or clone 22nucleic acid is separated from open reading frames which flank theIMP.18p or clone 22 gene and encode proteins other than IMP.18p or clone22. The term “purified” denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel. Particularly, itmeans that the nucleic acid or protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

[0050] As used herein, “nucleic acid,” “polynucleotide,” or “nucleicacid sequence” includes reference to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence includes the complementary sequencethereof. Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences andas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (see Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term encompasses nucleic acids, i.e., oligonucleotides,containing known analogues of natural nucleotides which have similar orimproved binding properties, for the purposes desired. The term alsoincludes nucleic acids which are metabolized in a manner similar tonaturally occurring nucleotides or at rates that are improved thereoverfor the purposes desired. The term also encompasses nucleic-acid-likestructures with synthetic backbones. DNA backbone analogues provided bythe invention include phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphor-amidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs);see Oligonucleotides and Analogues. A Practical Approach, edited by F.Eckstein, IRL Press at Oxford University Press (1991); AntisenseStrategies, Annals of the New York Academy of Sciences, Volume 600, Eds.Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.36:1923-1937; Antisense Research and Applications (1993, CRC Press) inits entirety and specifically Chapter 15, by Sanghvi, entitled“Heterocyclic base modifications in nucleic acids and their applicationsin antisense oligonucleotides.” PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed in WO 97/0321; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol144:189-197. Other synthetic backbones encompasses by the term includemethylphosphonate linkages or alternating methylphosphonate andphosphodiester linkages (Strauss-Soukup (1997) Biochemistry36:8692-8698), and benzylphosphonate linkages which, compared withunmodified oligonucleotides and methylphosphonates, are more stableagainst nucleases and exhibit a higher lipophilicity (Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acid isused interchangeably with gene, cDNA, mRNA, oligonucleotide primer,probe and amplification product. The term “exogenous nucleic acid”refers to a nucleic acid that has been isolated, synthesized, cloned,ligated, excised in conjunction with another nucleic acid, in a mannerthat is not found in nature, and/or introduced into and/or expressed ina cell or cellular environment other than or at levels or formsdifferent than the cell or cellular environment in which said nucleicacid or protein is be found in nature. The term encompasses both nucleicacids originally obtained from a different organism or cell type thanthe cell type in which it is expressed, and also nucleic acids that areobtained from the same cell line as the cell line in which it isexpressed, invention.

[0051] As used herein, “encoding” with respect to a specified nucleicacid, includes reference to nucleic acids which comprise the informationfor translation into the specified protein. The information is specifiedby the use of codons. Typically, the amino acid sequence is encoded bythe nucleic acid using the “universal” genetic code. However, variantsof the universal code, such as is present in some plant, animal, andfungal mitochondria, the bacterium Mycopiasma capricolum (Proc. Natl.Acad. Sci., 82:2306-2309 (1985), or the ciliate Macronucleus, may beused when the nucleic acid is expressed in using the translationalmachinery of these organisms.

[0052] As used herein, “having amino acid (or nucleic acid) sequenceidentity as measured using a sequence comparison algorithm” meansoptimal alignment of sequences for comparison using any means to analyzesequence identity (homology) known in the art, e.g., by the progressivealignment method of termed “PILEUP” (see below); by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981); by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970); by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85: 2444 (1988); by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.); or, by inspection. See also Morrison (1997) Mol. Biol.Evol. 14:428-441, as an example of the use of PileUp, ClustalW,TreeAlign, MALIGN, and SAM sequence alignment computer programs.

[0053] One example, PILEUP, creates a multiple sequence alignment from agroup of related sequences using progressive, pairwise alignments. Itcan also plot a tree showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences of a maximumlength of 5,000. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendopgram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison. For example, IMP.18p can be compared to other IMPsequences using the following parameters; default gap weight (3.00),default gap length weight (0.10), and weighted end gaps.

[0054] Another example of algorithm that is suitable for determiningsequence similarity is the BLAST algorithm, which is described inAltschul et al. J. Mol. Biol. 215: 403-410 (1990). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information, http://www.ncbi.nlm.nih.gov. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when; the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments, or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=-4, and a comparison of both strands.

[0055] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to IMP.18p nucleic acid ofSEQ ID NO:16 if the smallest sum probability in a comparison of the testnucleic acid to the IMP.18p nucleic acid is less than about 0.1, morepreferably less than about 0.01, and most preferably less than about0.00. Where the test nucleic acid encodes an IMP.18p or clone 22polypeptide, it is considered similar to the IMP.18p nucleic acid of SEQID NO:16 if the comparison results in a smallest sum probability of lessthan about 0.5, and more preferably less than about 0.2.

[0056] A “comparison window”, as used herein, includes reference to asegment of about 10 to 20 residues in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443; by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerizedimplementations of these algorithms (including, but not limited toCLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis. USA); the CLUSTAL program is well described by Higgins andSharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS 5:151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90;Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65,and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31.“Sequence identity” in the context of two nucleic acid or polypeptidesequences includes reference to the nucleotides (or residues) in the twosequences which are the same when aligned for maximum correspondenceover a specified “comparison window.” When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA). An indication that twopeptide sequences are substantially similar is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially similar to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

[0057] By “selectively hybridizing to,” “specifically hybridizing to” or“selective hybridization” is meant hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence to a detectably greater degree than itshybridization to non-target nucleic acid sequences. Specifically, asused herein, a specific or selective hybridization reaction (which is,by definition, under stringent hybridization conditions) will be atleast about 10 times greater than the background signal or noise.Generally, selectively hybridizing primer sequences yield an ampliconcomposition which can comprise at least 90% of the target amplicon.Selectively hybridizing sequences can have at least about 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other. “Percentage ofsequence identity” is determined by comparing two optimally alignedsequences over a comparison window (10-20 nucleotides), wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

[0058] As used herein, “stringent conditions” includes reference toconditions under which a nucleic acid sequence, such as a probe, willpreferentially hybridize to its target sequence and/or hybridize to itstarget sequence to the substantial exclusion of non-target sequences. Asdefined herein, a specific or selective hybridization reaction understringent hybridization conditions will be at least about 5 to 10 timesgreater than the background signal or noise. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 2×SSC at 50° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.“Stringent hybridization conditions” or “stringent conditions” in thecontext of nucleic acid hybridization assay formats are sequencedependent, and are different under different environmental parameters.An extensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York.

[0059] As used herein, “antibody composition” includes reference to atleast one antibody. In turn, “antibody” includes reference to animmunoglobulin molecule obtained by in vitro or in vivo generation ofthe humoral response, and includes both polyclonal and monoclonalantibodies. The term also includes genetically engineered forms such aschimeric antibodies (e.g., humanized murine antibodies), heteroconjugateantibodies (e.g., bispecific antibodies), and recombinant single chainFv fragments (scFv). The term “antibody” also includes antigen bindingforms of antibodies (e.g., Fab′, F(ab′)₂, Fab, Fv, rIgG, and, invertedIgG). See, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.). An antibody immunologically reactive with a particularantigen can be generated in vivo or by recombinant methods such asselection of libraries of recombinant antibodies in phage or similarvectors. See, e.g., Huse et al. (1989) Science 246:1275-1281; and Ward,et al. (1989) Nature 341:544-546; and Vaughan et al. (1996) NatureBiotechnology, 14:309-314.

[0060] As used herein, “specifically reactive” includes reference to thepreferential association of a ligand, in whole or part, with aparticular target molecule (i.e., “binding partner” or “binding moiety”)relative to compositions lacking that target molecule. As definedherein, a specific or selective binding reaction will be at least about10 times greater than the background signal or noise. It is, of course,recognized that a certain degree of non-specific interaction may occurbetween a ligand and a non-target molecule. Nevertheless, specificbinding, may be distinguished as mediated through specific recognitionof the target molecule. Typically specific binding results in a muchstronger association between the ligand and the target molecule thanbetween the ligand and non-target molecule. Specific binding by anantibody to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. The affinityconstant of the antibody binding site for its cognate monovalent antigenis at least between 10⁶-10⁷, usually at least 10⁸, preferably at least10⁹, more preferably at least 10¹⁰, and most preferably at least 10¹¹liters/mole. The phrase “specifically (or selectively) binds to anantibody” or “specifically (or selectively) immunoreactive with.” refersto an antibody binding reaction (including, at a minimum, an immunogenicbinding fragment) that is determinative of the presence of a protein ina heterogeneous population of proteins and other compositions orbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein and do not bind in a significantamount to other proteins present in the sample. As defined herein, aspecific or selective antibody binding reaction will be at least about10 times greater than the background signal or noise. Specific bindingto an antibody under such conditions may require an antibody that isselected for its specificity for a particular protein. For example,antibodies raised to IMP.18p with the amino acid sequence encoded in SEQID NO:17 are selected to obtain antibodies specifically immunoreactivewith IMP.18p proteins and polymorphic variants of IMP.18p within thescope of the claimed invention, and not with other proteins. Theanti-IMP.18p antibodies and antisera of the invention have less than 10%cross-reactivity to (e.g., as they are immunosorbed against) previouslycharacterized anti-IMP polypeptides, as discussed below.

[0061] A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein and itspolymorphic variants, as discussed in detail below. Solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunorcactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity). Forexample, as discussed below, a competitive binding immunoassay is usedto identify and isolate putative IMP.18p polymorphic variants within thescope of the claimed invention.

[0062] An “immunogen” or “immunogenic fragment” refers to a compound orcomposition comprising a carbohydrate, peptide, polypeptide or proteinwhich is “immunogenic,” i.e., capable of eliciting, augmenting orboosting a cellular and/or humoral immune response, either alone or incombination or linked or fused to another substance. An immunogeniccomposition can be a peptide of at least about 5 amino acids, a peptideof 10 amino acids in length, or preferably, the a fragment 15 aminoacids in length and more preferably a fragment 20 amino acids in lengthor greater. The immunogen (immunogenic fragment) can comprise a“carrier” polypeptide and a hapten (e.g., a carrier polypeptide fused orlinked (chemically or otherwise) to a peptide/protein fragment againstwhich the desired antibody will specifically recognize). The immunogencan be recombinantly expressed in an immunization vector, which can besimply naked DNA comprising the immunogen's coding sequence operablylinked to a promoter. The immunogen (immunogenic fragment) includesantigenic determinants, or epitopes (described below), to whichantibodies or TCRs bind, which are typically 3 to 10 amino acids inlength. An “immunological carrier” is an composition which, when linked,joined, chemically coupled or fused to a second composition (e.g.,protein, peptide, polysaccharide or the like) boosts or augments thecellular or humoral response to the composition. Any physiologicmechanism can be involved in this augmentation or boosting of the immuneresponse. An immunogenic carrier is typically a polypeptide linked orfused to a second composition of interest—the immunogenicfragment—comprising a protein, peptide or polysaccharide, where thecarrier stimulates a cellular (T cell mediated) immune response thatboosts or augments the humoral (B cell mediated, antibody-generating)immune response to the composition of interest. These secondcompositions can be “haptens,” which are typically defined as compoundsof low molecular weight that are not immunogenic by themselves, butthat, when coupled to carrier molecules, can elicit antibodies directedto epitopes on the hapten. Alternatively, an immunogenic fragment can belinked to a carrier simply to facilitate manipulation of the peptide inthe generation of the immune response (see, for example, Rondard (1997)Biochemistry 36:8962-8968). An “epitope” refers to an antigenicdeterminant or antigen site on the immunogenic fragment that interactswith an antibody or a T cell receptor (TCR). An “antigen” is a moleculeor composition that induces the production of an immune response. Anantibody or TCR binds to a specific conformational (possiblycharge-dependent) domain of the antigen, called the “antigenicdeterminant” or ‘epitope’ (TCRs bind the epitope in association with athird molecule, a major histocompatibility complex (MHC) protein).

[0063] The term “immunologically reactive conditions” refers to anyenvironment in which antibodies can bind to antigens, such as theIMP.18p of the invention or immunogenic fragments thereof. Theseconditions can be physiologic conditions similar to those seen in vivo,or, in vitro conditions compatible with antibody-antigen binding, suchas in an immunological binding assay.

[0064] As used herein, “polypeptide”, “peptide” and “protein” are usedinterchangeably and include reference to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The amino acids and analogs referred to herein aredescribed by shorthand designations as follows: Amino Acid NomenclatureName 3-letter 1 letter Alanine Ala A Arginine Mg R Asparagine Asn NAspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln QGlycine Gly G Histidine His H Homoserine Hse — Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Methionine sulfoxide Met (O) —Methionine Met (S-Me) — methylsulfonium Norleucine Nle — PhenylalaninePhe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp WTyrosine Tyr Y Valine Val V

[0065] Those of ordinary skill will readily understand that proteins ofthe present invention embrace minor variants of the isoforms of clone 22SEQ ID NO:3 and SEQ ID NO:4; and, IMP.18p proteins. Accordingly, thepresent invention embraces conservatively modified variants of the clone22 and IMP.18p proteins and substantially similar variants of clone 22and IMP.18p proteins. The following six groups each contain amino acidsthat are conservative substitutions for one another:

[0066] 1) Alanine (A), Serine (S), Threonine (T);

[0067] 2) Aspartic acid (D), Glutamic acid (E);

[0068] 3) Asparagine (N), Glutamine (O);

[0069] 4) Arginine (R), Lysine (K);

[0070] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0071] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0072] See also, Creighton (1984) Proteins W. H. Freeman and Company.

[0073] One of ordinary skill will recognize that individualsubstitutions, deletions or additions to a protein sequence whichalters, adds or deletes a single amino acid or a small percentage ofamino acids in the encoded sequence is a “conservatively modifiedvariant” where the alteration results in the substitution of an aminoacid with a chemically similar amino acid.

[0074] As used herein, “calculated molecular weight” of a polypeptide orpeptide is the molecular weight based on the polypeptide's or peptide'sdeduced amino acid sequence—the deduced translation product—as encodedby the corresponding nucleic acid. In contrast, the “apparent” molecularweight is measured, empirical value. The apparent molecular weight of aprotein can be determined by many different methods, all known to one ofskill in the art. Some methods of determination include: SDS gelelectrophoresis, native gel electrophoresis, molecular exclusionchromatography, zonal centrifugation, mass spectroscopy. Disparitybetween results of different techniques can be due to factors inherentin the technique. For example, native gel electrophoresis, molecularexclusion chromatography and zonal centrifugation depend on the size ofthe protein. The proteins that are cysteine rich can form many disulfidebonds, both intra- and intermolecular. SDS gel electrophoresis dependson the binding of SDS to amino acids present in the protein. Some aminoacids bind SDS more tightly than others, therefore, proteins willmigrate differently depending on their amino acid composition. Massspectroscopy and calculated molecular weight from the sequence in partdepend upon the frequency that particular amino acids are present in theprotein and the molecular weight of the particular amino acid. If aprotein is glycosylated, mass spectroscopy results will reflect theglycosvlation but a calculated molecular weight may not.

[0075] As used herein, “recombinant” includes reference to a proteinproduced using cells that do not have in their native form an endogenouscopy of the DNA able to express the protein. The cells produce therecombinant protein because they have been genetically altered by theintroduction of the appropriate isolated nucleic acid sequence. The termalso includes reference to a cell, or nucleic acid, or vector, that hasbeen modified by the introduction of a heterologous nucleic acid or thealteration of a native nucleic acid to a form not native to that cell,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise abnormally expressed, under expressed or not expressed at all.

[0076] As used herein, “encoding” with respect to a specified nucleicacid, includes reference to nucleic acids which comprise the informationfor translation into the specified protein. The information is specifiedby the use of codons. Typically, the amino acid sequence is encoded bythe nucleic acid using the “universal” genetic code. However, variantsof the universal code, such as is present in some plant, animal, andfungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Natl.Acad. Sci., 82:2306-2309 (1985), or the ciliate Macronucleus, may beused when the nucleic acid is expressed in using the translationalmachinery of these organisms.

[0077] As used herein, “immunologically cross-reactive” or“immunologically reactive” includes reference to an antigen which isspecifically reactive with an antibody which was generated using thesame (“immunologically reactive”) or different (“immunologicallycross-reactive”) antigen.

[0078] As used herein, “isoform” includes reference to a family offunctionally related proteins that differ in their amino acid sequencesbut are derived from the same nuclear transcript.

[0079] The term “modulator” refers to any synthetic or natural compoundor composition that can change in any way activity of protein of theinvention, including IMP.18p or clone 22 proteins. An modulator can bean agonist or an antagonist. A modulator can be, but is not limited to,any organic and inorganic compound; including, for example, smallmolecules, peptides, proteins, sugars, nucleic acids, fatty acids andthe like.

[0080] Method of Determining Increased Susceptibility toManic-Depressive Illness

[0081] The present invention is directed to a method for determining agenotype associated with increased susceptibility to manic-depressiveillness. The method comprises determining the genotype of a humanindividual diagnosed as manic-depressive. Methods of genotyping are wellknown to those of ordinary skill in the art. The genotype is determinedusing at least one polymorphic marker from within the region ofchromosome 18 localized by and including the markers D18S843 andD18S869, see FIG. 3. Other markers within this region and the forward(F) and reverse (R) primers for amplification of and subsequent use ofthese markers for mapping are shown in Table 1.

[0082] Primers for polymorphic markers within this region of chromosome18, including the markers D18S843 and D18S869, are publicly available onthe internet. See, for example, The Genome Database at URL:http://gdbwww.gdb.org/; National Center for Biotechnology Information atURL: http://www.ncbi.nim.nih.gov/SCIENCE96/(cited in Science, Oct. 25,1996, incorporated herein by reference); Cooperative Human LinkageCenter at URL: http://www.chlc.org/; and the Location Database at URL:http://cedar.genetics.soton.ac.uk/public_html/the information availablein each of these databases on the date of filing is incorporated hereinby reference. Primers and probes for markers are available from theATCC. See the latest ATCC Repository listing, for example, on-lineInternet or ATCC/NIH Repository Catalogue of Human and Mouse DNA Probesand Libraries, Eighth Edition 1995 (American Type Culture Collection,Rockville, Md.), incorporated herein by reference.

[0083] In preferred embodiments, genotyping within the interval ofchromosome 18 localized by markers D18S843 and D18S869 (see FIG. 3) isdetermined using one of the markers selected from the group consistingof the marker of clone 22, D18S1116, and D18S1150. In a particularlypreferred embodiment, the marker of clone 22 is used for determining thegenotype. Preferably, the genotype within the interval of D18S843 andD18S869 is determined using markers D18S153 ((also designated S53 inTable 3, below), D18S40 (also designated S40 in Table 3). D18S482,D18S71 (also designated S71 in Table 3), or D18S843. TABLE 3 Infantbrain derived cDNA clones mapping to chromosome 18. Clone Our InsertdhEST Insert GenBank Accession Number Gene EST Cytogenetic Number Size(kB) Size (kb) 5° 3° Homology* Homology* Bin 1 2.4 1.7 R51685 R51596HK63KDAP NA M 2 1.4 NA R61592 R61536 unknown EST64032 M 3 1.4 2.1 T77800R38384 HS63KDAP NA M 4 1.4 1.6 R56762 R56915 unknown unknown M 5 1.2 1.4H08437 H08745 unknown unknown S 6 1.5 1.5 R54360 R54361 unknown unknownS 7 1.4 1.9 T78290 R37939 MBP NA S 8 1.3 2.4 R20367 R43753 unknownunknown M 9 1.2 1.2 R18592 R41672 unknown EST197262 S 10 1.3 1.4 R18875R37298 HS63KDAP NA M 11 1.5 1.5 R34535 R49065 PTPRM NA A 12 1.9 2.0H17695 H17080 MBP NA S 13 1.7 1.9 R52596 R52541 unknown unknown L 14 1.82.0 R13520 R20642 unknown unknown A 15 1.4 NA R16321 R41398 unknownEST228925 M 16 1.7 NA H08970 H09539 unknown unknown S 17 1.6 2.1 R17799R43004 unknown EST64032 M 18 1.5 1.0 R22831 R46021 MBF NA S 19 2.0 2.8R14016 R39139 unknown unknown M 20 1.1 1.3 R11914 R39106 unknownEST197262 S 21 1.5 2.0 R19053 R44040 unknown EST228925 M 22 1.1 1.2R19445 R44696 unknown unknown C 23 1.1 1.2 T80229 R38716 unknownD135928E S 24 1.2 1.5 R35001 R49388 unknown EST91427 A 25 1.3 2.1 R17655R43373 unknown unknown M 26 1.0 1.2 R20441 R44144 unknown EST197262 S 271.3 1.4 R19332 R44600 unknown EST91427 A 28 1.8 NA H08354 H08355 unknownEST91427 A 29 1.8 1.7 none R39845 unknown unknown B 30 1.3 1.4 R52394R52395 unknown EST30984 M 31 1.1 1.3 H17749 H17636 GNAL NA B 32 1.2 1.2H06013 H05964 unknown EST91427 A 33 2.7 1.3 T74001 T87210 unknownunknown M 34 1.9 1.3 T80579 R38876 unknown unknown A 35 1.4 NA R60481R60245 unknown EST91427 A 36 1.2 NA R59504 R59505 unknown unknown K 371.9 2.1 R20248 R43704 unknown unknown B 38 1.1 1.1 H08492 H08770 unknownunknown M 39 1.6 1.7 H11689 H11600 unknown unknown G 40 1.7 1.3 R19498R43846 HUMKIAAN NA N 41 1.6 2.0 H17610 H17501 unknown unknown S 42 1.85.3 R17567 R42907 unknown EST91427 A 43 1.5 1.5 R20380 R43767 unknownunknown S 44 1.6 1.6 H17267 H17268 unknown EST91427 A 45 1.4 1.4 T80517R38994 PTPRM NA A 46 1.6 1.4 R20075 none MBP NA S 47 1.2 1.3 T66113T65029 unknown unknown S 48 1.3 1.3 R15279 none unknown EST91427 A

[0084] As will be recognized by those of skill, the complementarysequences of these primers may likewise be employed for amplifying orselectively hybridizing and detecting their target marker. Additionaltarget regions may be identified by walking from known chromosomemarkers as described above. Techniques for chromosome walking are wellknown in the art as described in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, 1989. Vectors which areoptimized for chromosome walking are commercially available (e.g.,lambda-DASH and lambda-FIX (Stratagene Cloning Systems, La Jolla,Calif.).

[0085] New markers may result from physical mapping of the intervaldefined by (flanked by) markers D18S843 and D18S869, see FIG. 3. In aparticularly preferred embodiment, the polymorphic marker of clone 22 isemployed. The polymorphic marker of clone 22 is a microsatellite markercomprising a trinucleotide repeat amplified by primers with thesequences set forth in SEQ ID NO:1 and SEQ ID NO:2 (Table 1). Allele 1and allele 2 comprise the two polymorphisms at the clone 22 locus. Thepolymorphism amplified by primers of SEQ ID NO:1 and SEQ ID NO:2 is atrinucleotide repeat consisting essentially of 10 GCT trinucleotides forallele 1 (SEQ ID NO:14), while the polymorphism amplified by theseprimers is a trinucleotide repeat consisting essentially of 9 GCTtrinucleotides for allele 2. The presence of allele 2 (SEQ ID NO:15) ofthe polymorphic marker indicates an increased susceptibility tomanic-depressive illness.

[0086] Markers from within the region localized by and including markersD18S843 and D18S869 are linked to a locus associated with susceptibilityto manic-depressive illness (bipolar disorder). Linkage disequilibriumbetween a polymorphism from this region and the appearance ofmanic-depressive illness provides a means of associating the appearanceof that polymorphism in an individual with an increased susceptibilityto manic-depressive illness. Consequently, a polymorphism exhibitinglinkage disequilibrium with the appearance of manic-depressive illnesscan be used as a standard against which an increased susceptibility tomanic-depressive illness can be determined for an individual whosedisease status is unknown.

[0087] In the present method, a statistically significant correlationbetween the presence of a particular polymorphism with the presence ofmanic-depressive illness in an individual allows for the determinationof the genotype(s) associated with increased or decreased susceptibilityto familial manic-depressive illness. In a preferred embodiment, thetransmission disequilibrium test (TDT) is employed to determine agenotype associated with increased susceptibility to manic-depressiveillness. See, Spieiman et al., Am. J. Hum. Gene., 52:506-516 (1993);Spielman and Ewen, Am. J. Hum. Gene., 59:983-989 (1996), both of whichare incorporated herein by reference. Briefly, the TDT considers parentswho are heterozygous for an allele associated with disease and evaluatesthe frequency with which that allele or its alternate is transmitted toaffected offspring.

[0088] The genotype of the tested individual can be convenientlydetermined with at least one polymorphic marker localized within thechromosomal region defined (flanked) by and including markers D18S43 andD18S869 (FIG. 3). Typically, the same marker or markers are used as indetermining the genotype associated with increased susceptibility tomanic-depressive illness. In a preferred embodiment, the polymorphicmarker is amplified by primers which selectively hybridize, understringent conditions, to the same nucleic acid sequences as primers ofSEQ ID NO:1 and SEQ ID NO:2 (Table 1).

[0089] Methods of amplifying sequences are well known to those ofordinary skill in the art. Amplification systems include the polymerasechain reaction (PCR) system, strand displacement amplification (SDA),see, e.g., Diagnostic Molecular Microbiology: Principles andApplications, Ed. D. H. Persing et al., American Society forMicrobiology, Washington, D.C.; ligase chain reaction (LCR) (Wu (1989)Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene89:117); transcription amplification (Kwoh Proc. Natl. Acad. Sci. USA.86:1173 (1989)); and, self-sustained sequence replication (Guatelli(1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicaseamplification and other RNA polymerase mediated techniques (e.g., NASBA,Cangene, Mississauga, Ontario); see Berger (1987) Methods Enzymol.152:307-316, Sambrook, and Ausubel, as well as Mullis (1987) U.S. Pat.Nos. 4,683,195 and 4,683,202; Arnheim (1990) C&EN 36-47; Lomell J. Clin.Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu(1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564. Methodsfor cloning in vitro amplified nucleic acids are described in Wallace,U.S. Pat. No. 5,426,039.

[0090] The PCR process is well-known in the art and is thus notdescribed in detail herein. For a review of PCR methods and protocols,see, e.g., Innis, et al, eds. PCR Protocols, A Guide to Methods andApplication (Academic Press, Inc. San Diego, Calif., 1990). PCR reagentsand protocols are also available from commercial vendors, such as RocheMolecular Systems. See, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;4,965,188, each of which is incorporated herein by reference. The firststep of each cycle of the PCR involves the separation of the nucleicacid duplex formed by the primer extension. Once the strands areseparated, the next step in PCR involves hybridizing the separatedstrands with primers that flank the target sequence. The primers arethen extended to form complementary copies of the target strands. Forsuccessful PCR amplification, the primers are designed so that theposition at which each primer hybridizes along a duplex sequence is suchthat an extension product synthesized from one primer, when separatedfrom the template (complement), serves as a template for the extensionof the other primer. The cycle of denaturation, hybridization, andextension is repeated as many times as necessary to obtain the desiredamount of amplified nucleic acid.

[0091] In a preferred embodiment of the PCR process, strand separationis achieved by heating the reaction to a sufficiently high temperaturefor a sufficient time to cause the denaturation of the duplex but not tocause an irreversible denaturation of the polymerase. Template-dependentextension of primers in PCR is catalyzed by a polymerizing agent in thepresence of adequate amounts of four deoxyribonucleotide triphosphates(typically DATP, dGTP, dCTP, and dTTP) in a reaction medium comprised ofthe appropriate salts, metal cations, and pH buffering system. Suitablepolymerizing agents are enzymes known to catalyze template-dependent DNAsynthesis. The methods of the present invention may be performed on awide variety of human cells including somatic cell hybrids, purifiednuclei, chromosomal preparations or nucleic acid sequences comprising amarker to a chromosomal region of the present invention. The cells maybe somatic or germline and from any time in gestation includingfertilized embryo or preimplantation blastocysts. Preferably, somaticcells are employed to avoid the possibility of meiotic recombinationevents between a marker and locus associated with susceptibility tomanic-depressive illness and to more readily allow determination of thegenotype for a homologous chromosome pair.

[0092] The methods of the present invention may conveniently bepracticed with markers which differ as to sequence or length, such asRFLPs (restriction fragment length polymorphisms) and microsatellitemarkers such as STRPs (short tandem repeat polymorphisms) or VNTRs(variable number tandem repeats). Generally, the sizes will bedetermined by standard gel electrophoresis techniques as described inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, 1989, and Polymeropoulos et al. Genomics. 12:492-496(1992). Polyacrylamide eel electrophoresis is particularly preferredbecause of its capability of high discrimination. Generally,autoradiography is employed to simultaneously visualize and identify themarkers.

[0093] Amplification of markers is generally performed with labelednucleotide bases that provide a means for identifying the amplifiedproduct following the procedure. Alternatively, labeled nucleic acidprimers can be employed as probes.

[0094] Probes can be used to selectively hybridize and detect andisolate a nucleic acid sequence (e.g., a cDNA or gene) of interest. Forexample, labeled probes can be used to detect RFLP markers which differin size after digestion with one or more restriction enzymes which havebeen separated, as by electrophoresis.

[0095] Where the nucleic acid encoding a clone 22 or IMP.18p protein isto be used as a nucleic acid probe, it is often desirable to label thenucleic acid with detectable labels. The labels may be incorporated byany of a number of means well known to those of skill in the art. Thelabel can be simultaneously incorporated during the amplificationprocedure in the preparation of the nucleic acids. Thus, for example,polymerase chain reaction (PCR) with labeled primers or labelednucleotides will provide a labeled amplification product. In anotherpreferred embodiment, transcription amplification using a labelednucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates alabel into the transcribed nucleic acids.

[0096] Alternatively, a label may be added directly to an originalnucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Means ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example nick translation or end-labeling (e.g.,with a labeled RNA) by phosphorylation of the nucleic acid andsubsequent attachment (ligation) of a nucleic acid linker joining thesample nucleic acid to a label (e.g., a fluorophore).

[0097] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate. Probes may be labeledwith visual labels such as photoluminescents, Texas red, rhodamine andits derivatives, red leuco dye and 3,3′,5,5°-tetramethylbenzidine (TMB),fluorescein and its derivatives, dansyl, umbelliferone and the like.Enzymes such as horse radish peroxidase, alkaline phosphatase, orequivalents can be used, especially in ELISAs. Magnetic beads,fluorescent dyes (e g., fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,⁴C, or ³²P), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beadsare also useful labeling means. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241.

[0098] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

[0099] Those of skill will recognize that polymorphic markers within theregion localized within and including D18S843 and D18S869 can beidentified by variations at the protein level when the polymorphismoccurs within a coding region. The present invention includes the use ofpolymorphisms which manifest themselves at both the nucleic acid andprotein sequence levels. Accordingly, means of distinguishingpolymorphisms include, but are not limited to, differences arising fromantigenicity, substrate specificity, or activity of encoded proteins.

[0100] Isolation of nucleic acids from biological samples for use in thepresent invention may be carried out by a variety of means well known inthe art. For example, see those described in Rothbart et al. 1989, inPCR Technology (Erlich ed., Stockton Press, New York) and Han et al.,1987, Biochemistry, 26:1617-1625. Kits are also commercially availablefor the extraction of high-molecular weight DNA for PCR. These kitsinclude Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim,Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL,Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell,Keene, N.H.), DNA Extraction Kit (Stratagene, La Jolla, Calif.),TurboGen Isolation Kit (Invitrogen, San Diego, Calif.), and the like.Use of these kits according to the manufacturer's instructions isgenerally acceptable for purification of DNA prior to practicing themethods of the present invention. In some case, the informative markermay be transcribed into RNA by the cells. In this instance, RNA may beused for amplification or for comparison between the tested individualand affected family member.

[0101] In another aspect, the present invention provides a method fordetermining an increased susceptibility to manic-depressive illness inan individual. Due to linkage disequilibrium the presence of allele 2(SEQ ID NO:15) of the clone 22 polymorphism appears more frequentlyamongst individuals in the U.S. population who have increasedsusceptibility to manic-depressive illness than individuals who lackthis allele. Consequently, the presence of allele of clone 22 is itselfdeterminative of an increased susceptibility to manic-depressiveillness. The tested individual may be a member of any racial or ethnicgroup, including, for example, individuals of European, African, orAsian descent. In preferred embodiments, the tested individual is ofEuropean descent. The method comprises determining the genotype of theindividual using the polymorphic marker of clone 22. The polymorphicmarker of clone 22 can be amplified with oligonucleotide primers whichamplify the same polymorphic marker as primers of SEQ ID NO:1 and SEQ IDNO:2. Use of such primers on a target comprising allele 1 yields thenucleic acid having the sequence shown in SEQ ID NO:14. The allele 1polymorphism consists of 10 trinucleotide (GCT) repeats. Use these sameprimers with a target nucleic acid of allele 2 yields the nucleic acidhaving the sequence shown in SEQ ID NO:15. The allele 2 polymorphismconsists of 9 trinucleotide (GCT) repeats. Thus, primers of the presentinvention will amplify the region of the trinucleotide repeatpolymorphism of clone 22. Those of skill will recognize that the primingof a target sequence is performed under stringent conditions such thatthe primers selectively hybridize to their target sequence. Preferably,the primers employed to amplify the polymorphism of clone 22 comprisethe sequence of SEQ ID NO:1 and SEQ ID NO:2. The primers of SEQ ID NO:1and SEQ ID NO:2 may comprise additional sequences to aid in suchprocesses as purification, labeling, or subcloning. The use ofadditional 5′ terminal sequences (i.e. tails) or 5′ labels is well knownto the skilled artisan.

[0102] Nucleic Acid and Protein Compositions

[0103] The invention provides for novel nucleic acids, and proteinsencoded therefrom, derived from a specific area of human chromosome 18.Genetic variations in this chromosomal region have been shown to beassociated with manic depressive illness, including bipolar disease,making these nucleic acids and proteins useful as diagnostic markers andtargets for preventive and therapeutic treatments. Specific embodimentsinclude novel nucleic acids and proteins identified as clone 22 andIMP.18p, both of which are encoded in this chromosome 18 region. Theseand other sequences within the region localized by and including markersD18S843 and D18S869. being linked to a locus associated withsusceptibility to manic-depressive illness, are also used as diagnosticmarkers in the invention. The invention provides for novel nucleic acidand antibody reagents used to identify and isolate these nucleic acidssequences and proteins, The invention also provides for characterizationand isolation of related species of clone 22 and IMP18.p using the novelreagents of the invention.

[0104] For example, one embodiment provides for a method for detectingthe presence of, and thereby isolating, a polynucleotide sequenceencoding at least a portion of an IMP.18p myo-inositol monophosphatasein a biological sample, comprising the steps of reacting a biologicalsample suspected of containing an IMP.18p nucleic acid with a probecomprising a nucleotide sequence of an IMP.18p, or a fragment thereof,capable of hybridizing to a myo-inositol monophosphatase-encodingnucleic acid from the biological sample. Embodiments which provide for ameans of detecting these novel nucleic acids or proteins thus alsoprovide means to diagnosing a myo-inositol monophosphatase-relatedconditions in a mammal. These methods comprise obtaining a cell ortissue sample from the mammal; determining the amount of an gene productin the cell or tissue; and comparing the amount of the gene product inthe cell or tissue with the amount in a healthy cell or tissue of thesame type; wherein a different amount of gene product in the sample fromthe mammal and the healthy cell or tissue is diagnostic of amyo-inositol monophosphatase-related condition.

[0105] On another embodiment, the invention provides for clone 22nucleic acid and protein encoded therefrom. The common subsequence ofthe native (naturally occurring) clone 22 mRNA transcript is shown inDNA form as SEQ ID NO:6. This common sequence is expressed with one oftwo different 5′ untranslated regions, SEQ ID NO:12 or SEQ ID NO:13. Thepresent invention includes isolated nucleic acids comprising the commonsequence, the 5′ untranslated regions of SEQ ID NO:12 and SEQ ID NO:13,and subsequences thereof.

[0106] Two isoforms of clone 22 proteins are provided herein. Thepresent invention includes these isolated proteins and subsequencesthereof. One isoform of a clone 22 protein has the amino acid sequenceshown in SEQ ID NO:3. The present invention provides isolated nucleicacids comprising a nucleic acid encoding the clone 22 protein of SEQ IDNO:3 and subsequences thereof. The present invention also providesisolated proteins comprising the amino acid sequence shown SEQ ID NO:3and subsequences thereof.

[0107] The second isoform of the clone 22 protein comprises the aminoacid sequence of SEQ ID NO:3 but lacks the amino acid sequence fromposition 113 to 130 (i.e., EGCLWPSDSAAPRLGASE) (SEQ ID NO:5). The secondisoform has the protein sequence shown in SEQ ID NO:4. The presentinvention includes isolated nucleic acids comprising a nucleic acidencoding the alternatively spliced clone 22 protein of SEQ ID NO:4 andsubsequences thereof. The present invention also provides isolatedproteins comprising the amino acid sequence shown in SEQ ID NO:4 andsubsequences thereof. Thus, the present invention provides nucleic acids(“clone 22 nucleic acids”) and proteins (“clone 22 proteins”) whichinclude both full-length and subsequences of isolated native nucleicacids and proteins of clone 22.

[0108] With the amino acid sequences of the clone 22 and IMP.18pproteins provided herein, one of skill can readily construct a varietyof clones containing nucleic acids which encode the same protein butvary in nucleic acid sequence due to the degeneracy of the genetic code.Cloning methodologies to accomplish these ends, and sequencing methodsto verify the sequence of nucleic acids are well known in the art.Examples of appropriate cloning and sequencing techniques, andinstructions sufficient to direct persons of skill through many cloningexercises are found in Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory (1989)),Methods in Enzymology, Vol. 152; Guide to Molecular Cloning Techniques(Berger and Kimmel (eds.), San Diego: Academic Press. Inc. (1987)), orCurrent Protocols in Molecular Biology, (Ausubel, et al. (eds.), GreenePublishing and Wiley-Interscience, New York (1987). Product informationfrom manufacturers of biological reagents and experimental equipmentalso provide information useful in known biological methods.

[0109] In some embodiments the isolated nucleic acids of the presentinvention comprise the sequence shown in SEQ ID NO:6 from nucleotide 116to 1033 (i.e., the sequence coding for the protein of SEQ ID NO:3); thisnucleic acid is identified herein as SEQ ID NO:7. In other embodimentsnucleic acids of the present invention comprise the sequence shown inSEQ ID NO:6 from nucleotide 116 to 1033 but lacking the sequence fromnucleotide 452 to 505 corresponding to the region from Glu 113 to Glu130 (i.e., lacking the region coding for the protein of SEQ ID NO:5);this nucleic acid is identified herein as SEQ ID NO:8.

[0110] A nucleic acid encoding the protein of SEQ ID NO:3 or SEQ ID NO:4can be amplified from human brain cDNA libraries using primers whichselectively hybridize, under stringent conditions, to the same nucleicacid sequence as primers of SEQ ID NO:9 and SEQ ID NO:10. Thus, forexample, isolated nucleic acids encoding the isolated proteins of SEQ IDNO:3 or SEQ ID NO:4 can be amplified using oligonucleotide primers whichselectively hybridize, under stringent conditions, to the same nucleicacid sequences of SEQ ID NO:7 and SEQ ID NO:8, respectively, as primersof SEQ ID NO:9 and SEQ ID NO:10.

[0111] The IMP.18p nucleic acid sequence (SEQ ID NO:16) and proteinsequence information (SEQ ID NO:17) can be used to design PCR primerswhich can be used to identify related IMP species, such as: SEQ ID NO:18and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; and, SEQ ID NO:22 andSEQ ID NO:23, can be used to directly amplify IMP species. The SEQ IDNO:18 (forward) and SEQ ID NO:19 (reverse) primer pair amplifies fulllength IMP.18p cDNA protein coding sequence: 5′-ATG AAG CCG AGC GGC GAGGAG-3′ (SEQ ID NO:18) 5′-CTT CTC ATC ATC CCG CCC ATA G-3′ (SEQ ID NO:19)

[0112] PCR primers such as SEQ ID NO:20 (forward, beginning at residuenumber 901, see FIG. 5B) and SEQ ID NO:21 (reverse, beginning residue1380) can also be used to directly amplify new IMP species or togenerate a DNA probe that would include mature protein coding region andmuch of the 3′ untranslated region, i.e., the poly-A attachment site.These primers, whether used to directly amplify new IMP species, useddirectly as probes, or used to generate (by PCR amplification) longerDNA probes, will also hybridize to a wide variety of different IMPspecies, especially those including IMP sequence variants that arebetter conserved in the 3′-untranslated region than in the matureprotein coding region: 5′-CTC GAC CTC ATG GCT TGC AGA G-3′ (SEQ IDNO:20) 5′-CTG AGA ACG ATC CGC TTT ATC-3′ (SEQ ID NO:21)

[0113] PCR primers such as SEQ ID NO:22 (forward primer) and SEQ IDNO:23 (reverse) can also be used to directly amplify new IMP species andisoforms or to generate a DNA probe that would include an internalsubset of IMP coding sequence. SEQ ID NO:22 and SEQ ID NO:23 primer pairamplifies an internal block of the coding sequence of IMP.18p protein.SEQ ID NO:22 and SEQ ID NO:23 correspond to coding sequence immediatelyupstream and downstream of motif A and motif B (discussed below),respectively (amino acids number 98 to 111 and 230 to 244, respectively,see FIG. 6; as numbered in FIG. 5B). As can be seen in FIG. 6, theseprimers correspond to relatively non-conserved IMP sequence: 5′-GTG TGTGCT CAC CCC GAC TGT-3′ (SEQ ID NO:22) 5′-CCC GAA GTG TCT ATC ACG ATG-3′(SEQ ID NO:23)

[0114] The subsequences of the isolated nucleic acids of the presentinvention are at least N nucleotides in length, where N is any one ofthe integers selected from the group consisting of from 15 to 900.Typically, the subsequences are at least 20 nucleotides in length,preferably at least 25 nucleotides in length, preferably at least 30nucleotides in length, and often at least 35.40, or 50 nucleotides inlength. The subsequences of the isolated proteins of the presentinvention are at least N′ amino acids in length, where N′ is any one ofthe integers from 5 to 300. The amino acid subsequences are derived fromcontiguous amino acids from the protein sequences of SEQ ID NO:3 or SEQID NO:4. The nucleic acid subsequences are derived from contiguousnucleotides from the nucleic acid sequences of SEQ ID NO:7 or SEQ IDNO:8. “Contiguous” with respect to a specified number of amino acidresidues or nucleotides, includes reference to a sequence of amino acidsor nucleotides, respectively, of the specified number from within thespecified reference sequence which has the identical order of aminoacids or nucleotides and the same adjacent amino acids or nucleotides asin the reference sequence.

[0115] The present invention also provides isolated mammalian proteinscomprising a clone 22 protein subsequence and an IMP.18p subsequence ofat least 10 contiguous amino acids, preferably at least 15 contiguousamino acids, more preferably at least 20 contiguous amino acids, andmost preferably at least 25, 30, 35, or 40 contiguous amino acids. Inthe case of clone 22, these amino acid sequences are from SEQ ID NO:3.In the case of IMP.18p, these amino acid sequences are from SEQ IDNO:17. The isolated mammalian proteins are immunologicallycross-reactive to an antibody composition that is generated from (e.g.,screened, synthesized, or elicited) and specifically reactive to aprotein immunogen of SEQ ID NO:3 and SEQ ID NO:17 for clone 22 andIMP.18p, respectively. The mammalian protein may be isolated from anynumber of mammals including: rat, mice, cattle, dog, pig, guinea pig, orrabbit, and most preferably a primate such as macaques, chimpanzees, orhumans.

[0116] The isolated clone 22 and IMP.18p proteins of the presentinvention can be constructed using standard recombinant or syntheticmethods. Solid phase synthesis of isolated proteins of the presentinvention of less than about 50 amino acids in length may beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp.3-284 in The Peptides: Analysis, Synthesis, Biology, Vol. 2, SpecialMethods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem.Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984). Detaileddescriptions of the procedures for solid phase synthesis of nucleicacids by phosphite-triester, phosphotriester and H-phosphonatechemistries are widely available. For example, the solid phasephosphoramidite triester method of Beaucage and Carruthers using anautomated synthesizer is described in Itakura, U.S. Pat. No. 4,401,796;Carruthers, U.S. Pat. Nos. 4,458,066 and 4,500,707; Carruthers (1982)Genetic Engineering 4:1-17; see also Needham-VanDevanter (1984) NucleicAcids Res. 12:6159-6168; Beigelman (1995) Nucleic Acids Res 23:3989-3994; Jones, chapt 2, Atkinson, chapt 3, and Sproat, chapt 4, inOLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, Gait (ed.), IRL Press,Washington D.C. (1984); Froehler (1986) Tetrahedron Lett. 27:469472;Froehier, Nucleic Acids Res. 14:5399-5407 (1986); Sinha, TetrahedronLett. 24:5843-5846 (1983); and Sinha, Nucl. Acids Res. 12:4539-4557(1984). Methods to purify oligonucleotides include native acrylamide gelelectrophoresis, anion-exchange HPLC, as described in Pearson (1983) J.Chrom. 255:137-149. The sequence of the synthetic oligonucleotide can beverified using any chemical degradation method, for example, see Maxam(1980) Methods in Enzymology 65:499-560, Xiao (1996) Antisense NucleicAcid Drug Dev 6:247-258, or for solid-phase chemical degradationprocedures, Rosenthal (1987) Nucleic Acids Symp Ser 18:249-252.

[0117] Proteins of greater length may be synthesized by condensation ofthe amino and carboxy termini of shorter fragments. Methods of formingpeptide bonds by activation of a carboxy terminal end (e.g., by the useof the coupling reagent N,N′-dicycylohexyl carbodiimide) is known tothose of skill.

[0118] Subsequences of nucleic acids can be used as probes to detect orisolate the clone 22 and IMP.18p encoding nucleic acids for furtheranalysis of the polymorphism contained therein for purposes describedmore fully, supra. Additionally, subsequences can be utilized as primersfor amplification of the clone 22 and IMP.18p polymorphisms. Thesubsequence may be derived from within any portion of the clone 22isoforms and IMP.18p coding sequence. Probes specific to one or theother isoform of clone 22 can be used to study differentialtranscription of these isoforms.

[0119] Isolated nucleic acids of the present invention can also be usedfor recombinant expression of the proteins of the present invention foruse as immunogens in the preparation of antibodies. Subsequences canalso be used for detecting and/or quantifying clone 22 protein andIMP.18p expression by assaying for the gene transcript (e.g., nuclearRNA, mRNA) using nucleic acids coding for clone 22 and IMP.18p proteins.The assay can be for the presence or absence of the normal gene or geneproduct, for the presence or absence of an abnormal gene or gene productor quantification of the transcription levels of normal or abnormalclone 22 and IMP.18p gene product. Nucleic acid assays are well known inthe art and included in standard molecular biology references such asthose incorporated by reference herein.

[0120] For example, amongst the various hybridization formats well knownto the skilled artisan is included solution phase, solid phase, mixedphase, or in situ hybridization assays. Briefly, in solution (or liquid)phase hybridizations, both the target nucleic acid and the probe orprimer are free to interact in the reaction mixture. In solid phasehybridization assays, probes or primers are typically linked to a solidsupport where they are available for hybridization with target nucleicin solution. In mixed phase, nucleic acid intermediates in solutionhybridize to target nucleic acids in solution as well as to a nucleicacid linked to a solid support. In in situ hybridization, the targetnucleic acid is liberated from its cellular surroundings in such as tobe available for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the various hybridizationassay formats: Singer et al., Biotechniques 4(3):230-250 (1986); Haaseet al., Methods in Virology, Vol. VII, pp. 189-226 (1984); Wilkinson,“The theory and practice of in situ hybridization” In: In situHybridization, Ed. D. G. Wilkinson. IRL Press, Oxford University Press,Oxford; and Nucleic Acid Hybridization: A Practical Approach, Ed, Hames,B. D. and Higgins, S. J., IRL Press (1987).

[0121] Those of skill in the art will appreciate that various degrees ofstringency of hybridization can be employed in the assay; and either thehybridization or the wash medium can be stringent. A %s the conditionsfor hybridization become more stringent, there must be a greater degreeof complementarity between the probe and the target for duplex formationto occur. The degree of stringency can be controlled by temperature,ionic strength, pH and the presence of a partially denaturing solventsuch as formamide. For example, the stringency of hybridization isconveniently varied by changing the polarity of the reactant solutionthrough manipulation of the concentration of formamide within the rangeof 0% to 50%.

[0122] The degree of complementarity (sequence identity) required fordetectable binding will vary in accordance with the stringency of thehybridization medium and/or wash medium. The degree of complementaritywill optimally be 100 percent; however, it should be understood thatminor sequence variations in the probes and primers may be compensatedfor by reducing the stringency of the hybridization and/or wash mediumas described below. Thus, despite the lack of 100 percentcomplementarity under reduced conditions of stringency, functionalnucleic acids of the present invention having minor base differencesfrom the nucleic acid targets are possible. Therefore, underhybridization conditions of reduced stringency, it may be possible toconstruct an oligonucleotide having substantial identity to anoligonucleotide complementary to the target sequence while maintainingan acceptable degree of specificity. Substantial identity in the contextof nucleic acids means that the two molecules hybridize to each otherunder stringent conditions. Generally, stringent conditions are selectedto be about 5° C. to 200 C lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. Typically,stringent conditions will be those in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least about 60° C.more preferably 65° C.; however, for in situ hybridization thetemperature is preferably 40° C. Stringent conditions typically includeat least one wash in 0.2×SSC at a temperature of at least about 50° C.,usually about 55° C. to about 60° C., for 20 minutes, or equivalentconditions. The hybridization format or buffers are not critical aspectsof the present invention and those of skill will recognize that furtheradvances, improvements, or modifications in nucleic acid hybridization,amplification, and detection are within the scope of the invention.

[0123] The nucleic acids of the present invention, whether derived froma biological source, artificially constructed or both, can be operablylinked to a promoter. Those of ordinary skill will recognize that anisolated duplex clone 22 or IMP.18p nucleic acid operably linked to apromoter in forward orientation can direct transcription of mRNA whichcan be translated into a clone 22 or IMP.18p protein of the presentinvention. An isolated duplex clone 22 or IMP.18p nucleic acid operablylinked to a promoter in reverse orientation can direct transcription ofantisense mRNA. Antisense nucleic acids can be used for probes in assaysfor normal or abnormal gene product or to quantitate the expression ofmRNA coding for the clone 22 or IMP.18p protein in, for example, drugassays. Accordingly, the isolated nucleic acids of the present inventionare inclusive of both sense and antisense nucleic acids.

[0124] The isolated nucleic acid compositions of this invention, whetherRNA, cDNA, genomic DNA, or a hybrid of the various combinations, areisolated from biological sources or synthesized in vitro.Deoxynucleotides encoding isolated proteins of the present invention canbe prepared by any suitable method including, for example, cloning andrestriction of appropriate sequences as discussed supra, or by directchemical synthesis by methods such as the phosphotriester method ofNarang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester methodof Brown et al., Meth. Enzymol. 68: 109-151 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862 (1981); the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981), Tetrahedron Letts.,22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,12:6159-6168; and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0125] Once the nucleic acid encoding a protein of the present inventionis isolated and cloned, one may express the desired protein in arecombinantly engineered cell such as bacteria, yeast, insect(especially employing baculoviral vectors), and mammalian cells. It isexpected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteins. Noattempt to describe in detail the various methods known for theexpression of proteins in prokaryotes or eukaryotes will be made. Inbrief, the expression of natural or synthetic nucleic acids encoding theisolated proteins of the invention will typically be achieved byoperably linking the DNA or cDNA to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encodingthe protein. To obtain high level expression of a cloned gene, it isdesirable to construct expression vectors which contain, at the minimum,a strong promoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.One of skill would recognize that minor modifications can be made to aclone 22 or IMP.18p protein. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, a methionine addedat the amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

[0126] Examples of techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates.Inc. and John Wiley & Sons, Inc. (1994 Supplement) (Ausubel); Cashion etal. U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.Cloning vectors and host cells are readily obtained through commercialsources or from the American Type Culture Collection, each of which isincorporated herein by reference.

[0127] 1. Expression in Prokaryotes

[0128] Bacterial strains which can be used to express the nucleic acidof the invention include Escherichia coli, Bacillus subtillus,Streptococcus cremoris, Streptococcus lactis, Streprococcusthermophilus, Leuconostoc citrovorum, Leuconostoc mesenteroides,Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacteriumbifidum, Bifidobacteriu breve, and Bifidobacterium longum.

[0129] Examples of regulatory regions suitable for this purpose in E.coli are the promoter and operator region of the E. coli tryptophanbiosynthetic pathway as described by Yanofsky, Bacteriol. 158:1018-1024(1984), and the leftward promoter of phage lambda (P_(L)) as describedby Herskowitz and Hagen. Ann Rev. Gene., 14:399-445 (1980). Theinclusion of selection markers in DNA vectors transfected in E. coli isalso useful. Examples of such markers include genes specifyingresistance to ampicillin, tetracycline, or chloramphenicol. See,Sambrook, et al. for details concerning selection markers for use in E.coli.

[0130] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for clone 22 proteins are available using E. coli,Bacillus sp. and Salmonella (Palva, et al., Gene 22:229-235 (1983);Mosbach, et al., Nature 302:543-545 (1983)).

[0131] When expressing clone 22 or IMP.18p proteins in S. typhimurium,one should be aware of the inherent instability of plasmid vectors. Tocircumvent this, the foreign gene can be incorporated into anonessential region of the host chromosome. This is achieved by firstinserting the gene into a plasmid such that it is flanked by regions ofDNA homologous to the insertion site in the Salmonella chromosome. Afterintroduction of the plasmid into the S. typhimurium, the foreign gene isincorporated into the chromosome by homologous recombination between theflanking sequences and chromosomal DNA.

[0132] An example of how this can be achieved is based on the his operonof Salmonella. Two steps are involved in this process. First, a segmentof the his operon must be deleted in the Salmonella strain selected asthe carrier. Second, a plasmid carrying the deleted his regiondownstream of the gene encoding the clone 22 or IMP.18p protein istransfected into the his Salmonella strain. Integration of both the hissequences and a gene encoding a clone 22 or IMP.18p protein occurs,resulting in recombinant strains which can be selected as his⁺.

[0133] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Bacteria are grown according to standard procedures in theart. Because some proteins can be difficult to isolate with intactbiological activity, preferably fresh bacteria cells are used forisolation of protein. Use of cells that are frozen after growth butprior to lysis typically results in negligible yields of active protein.

[0134] Detection of the expressed protein is achieved by methods knownin the art and include, for example, radioimmunoassays, Western blottingtechniques or immunoprecipitation.

[0135] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, anon-ionic detergent. The cell suspension can be homogenized using aPolytron (Brinkman Instruments, Westbury, N.Y.). Alternatively, thecells can be sonicated on ice. Alternate methods of lysing bacteria areapparent to those of skill in the art (see, e.g., Sambrook et al.,supra: Ausubel et al., supra).

[0136] The cell suspension is generally centrifuged and the pelletcontaining the inclusion bodies resuspended in buffer that does notdissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2),1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. Itmay be necessary to repeat the wash step to remove as much cellulardebris as possible. The remaining pellet of inclusion bodies may beresuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH6.8, 150 mM NaCl). Other appropriate buffers will be apparent to thoseof skill in the art.

[0137] Following the washing step, the inclusion bodies are solubilizedby the addition of a solvent that is both a strong hydrogen acceptor anda strong hydrogen donor (or a combination of solvents each having one ofthese properties); the proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventswhich are capable of solubilizing aggregate-forming proteins for exampleSDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. After solubilization, the protein can se separated from otherbacterial proteins by standard separation techniques.

[0138] Alternatively, it is possible to purify the protein of interestfrom bacteria periplasm. Where IMP.18p or clone 22, for example, isexported into the periplasm of the bacteria, the periplasmic fraction ofthe bacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art. Purification from E. coli can alsobe achieved following procedures described in U.S. Pat. No. 4,511,503.

[0139] 2. Expression in Eukaryotes

[0140] A variety of eukaryotic expression systems such as yeast, insectcell lines, bird, fish, frog, and mammalian cells, are known to those ofskill in the art. As explained briefly below, the isolated proteins ofthe present invention may be expressed in these eukaryotic systems.

[0141] Yeast expression systems being eukaryotic, provide an attractivealternative to bacterial systems for some applications, for an overviewof yeast expression systems, see, Protein Engineering Principles andPractice, eds. Cleland et al. Wiley-Liss, Inc. p 129 (1996). A varietyof yeast vectors are publicly available. For example, the expressionvector pPICZ B (Invitrogen, San Diego, Calif.) can be used to expressthe protein of the invention in yeast, such as Picihia pastoris. Yeastepisomal plasmids comprising inducible promoters can be used for theintracellular expression of proteins the invention. Vectors include thepYES2 expression vector (Invitrogen. San Diego, Calif.) and pBS24.1(Boeke (1984) Mol. Gen. Gene. 197:345); see also Jacobs (1988) Gene67:259-269. Yeast promoters for yeast expression vectors suitable forexogenous protein expression include the inducible promoter from thealcohol dehydrogenase gene. AADH2, also called the yeast alcoholdehydrogenase II gene promoter (ADH2P). The protein of interest can befused at the amino terminal end to the secretion signal sequence of theyeast mating pheromone alpha-factor (MF alpha 1S) and fused at thecarboxy terminal end to the alcohol dehydrogenase II gene terminator(ADH2T), see van Rensburg (1997) J. Biotechnol. 55:43-53. The yeastalpha mating pheromone signal sequence allows for secretion of theexpressed polypeptide. Direct intracellular expression of IMP.18p isuseful for a variety of cell-based screens for activity and modulatorsof enzyme activity.

[0142] Yeast strains which can be used to express exogenous nucleicacids include Pichia pastoris, Hansenula polymorpha, Torulopsis holmil,Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis,and Candida pseudotropicalis. A large number of vectors are availablefor S. cerevisiae. Kluyveromyces lactis, and the methylotrophs Hansenulapolymorpha s and Pichia pastoris offer certain advantages over baker'syeast S. cerevisiae for the production of certain proteins, seeGellissen (1997) Gene 190:87-97; Wegner (1990) FEMS Microbiol. Rev.87:279.

[0143] Synthesis of heterologous proteins in yeast is well known.Methods in Yeast Genetics, Sherman, F., et al., Cold Spring HarborLaboratory, (1982) is a well recognized work describing the variousmethods available to produce the protein in yeast. Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Forinstance, suitable vectors are described in the literature (Botstein, etal., 1979, Gene, 8:17-24; Broach, et al., (1979), Gene, 8:121-133).

[0144] Two procedures are used in transfecting yeast cells. In one case,yeast cells are first converted into protoplasts using zymolyase,lyticase or glusulase, followed by addition of DNA and polyethyleneglycol (PEG). The PEG-treated protoplasts are then regenerated in a 3%agar medium under selective conditions. Details of this procedure aregiven in the papers by J. D. Beggs, (1978), Nature (London),275:104-109; and Hinnen, A., et al. (1978), Proc. Natl. Acad. Sci. USA,75:1929-1933. The second procedure does not involve removal of the cellwall. Instead the cells are treated with lithium chloride or acetate andPEG and put on selective plates (Ito, H., et al. (1983), J. Bact.,153:163-168).

[0145] Clone 22 proteins or IMP.18p, once expressed, can be isolatedfrom yeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayof other standard immunoassay techniques.

[0146] The sequences encoding clone 22 or IMP.18p proteins can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, bird, amphibian, or fishorigin. Illustrative of cell cultures useful for the production of thepeptides are mammalian cells. Mammalian cell systems often will be inthe form of monolayers of cells although mammalian cell suspensions mayalso be used. A number of suitable host cell lines capable of expressingintact proteins have been developed in the art, and include the CHO celllines, and various human cells such as COS cell lines, HeLa cells,myeloma cell lines, Jurkat cells. Other animal cells useful forproduction of IMP18.p and clone 22 proteins are available, for instance,from the American Type Culture Collection Catalogue of Cell Lines andHybridomas (7th edition. 1992).

[0147] Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g., the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. The expression vectortypically contains a transcription unit or “expression cassette” thatcontains all the additional elements required for the expression of theIMP18.p or clone 22 encoding DNA in host cells. A typical expressioncassette thus contains a promoter operably linked to the DNA sequenceencoding protein coding sequence and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination.

[0148] The DNA sequence encoding the IMP18.p and clone 22 proteins cantypically be linked to a cleavable signal peptide sequence to promotesecretion of the encoded protein by the transformed cell. Such signalpeptides would include, among others, the signal peptides from tissueplasminogen activator, insulin, and neuron growth factor, and juvenilehormone esterase of Heliothis virescens.

[0149] Additional elements of the expression cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

[0150] Appropriate vectors for expressing clone 22 or IMP.18p proteinsin insect cells arc usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider J.Embryol. Exp. Morphol. 27:353-365 (1987).

[0151] As indicated above, the vector, e.g., a plasmid which is used totransfect the host cell, preferably contains DNA sequences to initiatetranscription and sequences to control the translation of the protein.These sequences are referred to as expression control sequences.

[0152] As with yeast, when higher animal host cells are employed,polyadenlyation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenlyation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, J. et al., (1983), J. Virol. 45: 773-781).

[0153] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein Barvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.Additionally, gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus type-vectors. Saveria-Campo, M., 1985. “Bovine Papilloma virus DNAa Eukaryotic Cloning Vector” in DNA Cloning Vol. II a Practical ApproachEd. D. M. Glover, IRL Press, Arlington, Va. pp. 213-238.

[0154] Some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. Gene amplification, whether by highervector copy number or by replication of a gene in a chromosome, canincrease yields of recombinant proteins in mammalian and other cells.One in vitro amplification method for heterologous gene expression inmammalian cells is based on the stable transfection of cells with long,linear DNA molecules having several copies of complete expression units,coding for the gene of interest, linked to one terminal unit coding fora selectable marker. As another example, gene amplification of the geneof interest can be achieved by linking it to a dihydrofolate reductase(Dhfr) gene and administering methotrexate to the transfected cells,this method can increase recombinant protein production many fold (seeMonaco (1996) Gene 180:145-150).

[0155] Alternatively, high yield expression systems not involving geneamplification are also suitable, such as using a bacculovirus vector ininsect cells, with for example an IMP.18p encoding sequence under thedirection of the polyhedrin promoter or other strong baculoviruspromoters. A commonly used insect system utilizes Spodoptera frugiperdainfected with a baculovirus, such as Autographa californica nuclearpolyhedrosis virus. This virus can be used to infect Sf21 (Deutschmann(1994) Enzyme Microb Technol 16:506-512) or Sf9 cells (MaxBac 2.0,Invitrogen, San Diego, Calif.) (Zhu (1996) J Virol Methods 62(1), 71-79)derived from Spodoptera frugiperda, High Five cells derived fromTrichoplusia ni insect cells (Parrington (1997) Virus Genes 14(1),63-72), and Lymantria dispar (Vaughn (1997) In Vitro Cell Dev Biol Minim33:479-482); see also Grabherr (1997) Biotechniques 22: 730-735).Baculovirus transfer vectors can be used to replace the wild-type AcMNPVpolyhedron gene with a heterologous gene of interest. Sequences thatflank the polyhedrin gene in the wild-type genome are positioned 5′ and3′ of the expression cassette on the transfer vectors. Followingcotransfection with AcMNPV DNA, a homologous recombination event occursbetween these sequences resulting in a recombinant virus carrying thegene of interest and the polyhedrin or p10 promoter. Baculovirusexpression vectors are publicly available, such as pAC360 (Invitrogen,San Diego, Calif.). In addition to manufacturer s instructionsaccompanying the commercially available baculovirus systems, see“Current Protocols in Molecular Biology,” Ausubel, Chapter 16.

[0156] The host cells are competent or rendered competent fortransfection by various means. There are several well-known methods ofintroducing DNA into animal cells. These include: calcium phosphateprecipitation, fusion of the recipient cells with bacterial protoplastscontaining the DNA, treatment of the recipient cells with liposomescontaining the DNA, DEAE dextran, electroporation and micro-injection ofthe DNA directly into the cells. The transfected cells are cultured bymeans well known in the art. Biochemical Methods in Cell Culture andVirology, Kuchler, R. J. Dowden, Hutchinson and Ross, Inc., (1977). Theexpressed proteins are recovered by well known mechanical, chemical orenzymatic means.

[0157] The clone 22 or IMP.18p proteins of the present invention whichare produced by recombinant DNA technology may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced clone 22 or IMP.18p proteins can be directly expressed orexpressed as a fusion protein. The recombinant clone 22 or IMP.18pprotein can be purified by a combination of cell lysis (e.g.,sonication) and affinity chromatography. For fusion products, subsequentdigestion of the fusion protein with an appropriate proteolytic enzymereleases the desired recombinant clone 22 or IMP.18p protein.

[0158] The clone 22 or IMP.18p proteins of this invention, recombinantor synthetic, may be purified to substantial purity by standardtechniques well known in the art, including selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press, 1990.For example, antibodies may be raised to the clone 22 or IMP.18pproteins as described herein. The protein may then be isolated fromcells expressing the recombinant clone 22 or IMP.18p protein and furtherpurified by standard protein chemistry techniques as described above.

[0159] Antibodies

[0160] The present invention provides antibodies specifically reactive,under immunologically reactive conditions, to an isolated protein of thepresent invention. Antibodies are raised to a protein of the presentinvention, including individual, allelic strain, or species variants andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated.

[0161] Many methods of making antibodies are known to persons of skill.The following discussion is presented as a general overview of thetechniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

[0162] A. Antibody Production

[0163] A number of immunogens are used to produce antibodiesimmunologically reactive with a clone 22 or IMP.18p protein. An isolatedrecombinant, synthetic, or native clone 2e protein of 5 contiguous aminoacids in length or greater from SEQ ID NO:3 or 4 is the preferredimmunogens (antigen) for the production of anti-clone 22 polypeptidemonoclonal or polyclonal antibodies. An isolated recombinant, synthetic,or native IMP.18p protein of 5 contiguous amino acids in length orgreater from SEQ ID NO:17 is the preferred immunogens (antigen) for theproduction of anti-IMP.18p polypeptide monoclonal or polyclonalantibodies. In one class of preferred embodiments, an immunogenicprotein conjugate is also included as an immunogen. Naturally occurringclone 22 or IMP.18p proteins are also used either in pure or impureform.

[0164] The clone 22 or IMP.18p protein is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the clone 22 or IMP.18p protein.Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen (antigen), preferably a purifiedclone 22 or IMP.18p protein, a clone 22 or IMP.18p protein coupled to anappropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or aclone 22 or IMP.18p protein incorporated into an immunization vectorsuch as a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to the clone22 or IMP.18p protein of interest. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the clone 22 or IMP.18p protein isperformed where desired (see, e.g., Coligan (1991) Current Protocols inImmunology Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Harbor Press, NY).

[0165] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments of clone22 or IMP.18p protein are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a clone 22 or IMP.18p protein ofat least about 5 amino acids, more typically the clone 22 or IMP.18pprotein is at least 10 amino acids in length, preferably, at least 15amino acids in length, more preferably at least 25 amino acids inlength. In particularly preferred embodiments, the immunogen is derivedfrom the extra- or intra-cytoplasmic region of the clone 22 protein. Thepeptides are typically coupled to a carrier protein (e.g., as a fusionprotein), or are recombinantly expressed in an immunization vector.Antigenic determinants on peptides to which antibodies bind aretypically 3 to 10 amino acids in length.

[0166] Monoclonal antibodies are prepared from cells secreting thedesired antibody. Monoclonals antibodies are screened for binding to aclone 22 or IMP.18p protein from which the immunogen was derived.Specific monoclonal and polyclonal antibodies will usually bind with anaffinity constant of at least between 10⁻⁶ to 10⁻⁷ M, preferably atleast 10⁻⁸ M, preferably at least 10⁻⁹ M, more preferably at least 10⁻¹⁰M, most preferably at least 10⁻¹¹ M.

[0167] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animalwith an immunogen comprising a clone 22 or IMP.18p protein. The animalis then sacrificed and cells taken from its spleen, which are fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

[0168] Alternative methods of immortalization include transfection withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells is enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host. The clone22 or IMP.18p proteins and antibodies of the present invention are usedwith or without modification, and include chimeric antibodies such ashumanized murine antibodies.

[0169] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phase or similar vectors (see, e.g., Huse etal. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology, 14: 309-314).Alternatively, high avidity human monoclonal antibodies can be obtainedfrom transgenic mice comprising fragments of the unrearranged humanheavy and light chain Ig loci (i.e., minilocus transgenic mice).Fishwild et al. Nature Biotech., 14:845-851 (1996).

[0170] Frequently, the clone 22 or IMP.18p proteins and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

[0171] The antibodies of this invention are also used for affinitychromatography in isolating clone 22 or IMP.18p protein. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified clone 22 orIMP.18p protein are released.

[0172] The antibodies can be used to screen expression libraries forparticular expression products such as normal or abnormal human clone 22or IMP.18p proteins. Usually the antibodies in such a procedure arelabeled with a moiety allowing easy detection of presence of antigen byantibody binding.

[0173] Antibodies raised against a clone 22 or IMP.18p protein can alsobe used to raise anti-idiotypic antibodies. These are useful fordetecting or diagnosing various pathological conditions related to thepresence of the respective antigens.

[0174] B. Human or Humanized (Chimeric) Antibody Production

[0175] The anti-clone 22 or anti-IMP.18p protein antibodies of thisinvention can also be administered to a mammal (e.g., a human patient)for therapeutic purposes (e.g., as targeting molecules when conjugatedor fused to effector molecules such as labels, cytotoxins, enzymes,growth factors, drugs, etc.). Antibodies administered to an organismother than the species in which then, are raised are often immunogenic.Thus, for example, murine antibodies administered to a human ofteninduce an immunologic response against the antibody (e.g., the humananti-mouse antibody (HAMA) response) on multiple administrations. Theimmunogenic properties of the antibody are reduced by altering portions,or all, of the antibody into characteristically human sequences therebyproducing chimeric or human antibodies, respectively.

[0176] i) Humanized (Chimeric) Antibodies

[0177] Humanized (chimeric) antibodies are immunoglobulin moleculescomprising a human and non-human portion. More specifically, the antigencombining region (or variable region) of a humanized chimeric antibodyis derived from a non-human source (e.g., murine) and the constantregion of the chimeric antibody (which confers biological effectorfunction to the immunoglobulin) is derived from a human source. Thehumanized chimeric antibody should have the antigen binding specificityof the non-human antibody molecule and the effector function conferredby the human antibody molecule. A large number of methods of generatingchimeric antibodies are well known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856,5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238,5,169,939, 5,081,235, 5,075,431, and 4,975,369). Detailed methods forpreparation of chimeric (humanized) antibodies can be found in U.S. Pat.No. 5,482,856.

[0178] ii) Human Antibodies

[0179] In another embodiment, this invention provides for fully humananti-clone 22 or anti-IMP.18p protein antibodies. Human antibodiesconsist entirely of characteristically human polypeptide sequences. Thehuman anti-clone 22 or anti-IMP.18p protein antibodies of this inventioncan be produced in using a wide variety of methods (see, e.g., Larricket al., U.S. Pat. No. 5,001,065, for review).

[0180] In preferred embodiments, the human anti-clone 22 or anti-IMP.18pprotein antibodies of the present invention are usually producedinitially in trioma cells. Genes encoding the antibodies are then clonedand expressed in other cells, particularly, nonhuman mammalian cells.The general approach for producing human antibodies by trioma technologyhas been described by Ostberg et al. (1983), Hybridoma 2: 361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells: two humanand one mouse. Triomas have been found to produce antibody more stablythan ordinary hybridomas made from human cells.

[0181] The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingthe polymerase chain reaction, known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152:Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego,Calif., 1987; Co et al. (1992) J. Immunol., 148: 1149). For example,genes encoding heavy and light chains are cloned from a trioma's genomicDNA or cDNA produced by reverse transcription of the trioma's RNA.Cloning is accomplished by conventional techniques including the use ofPCR primers that hybridize to the sequences flanking or overlapping thegenes, or segments of genes, to be cloned.

[0182] Clone 22 and IMP.18p Protein Immunoassays

[0183] Embodiments include means of detecting the clone 22 or IMP.18pproteins of the present invention using novel reagents provided for bythe invention. In one embodiment, the clone 22 or IMP.18p proteins aredetected and/or quantified using the novel antibodies provided for bythe invention utilizing any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr, eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this caseclone 22 or IMP.18p protein). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds a clone 22 orIMP.18p protein(s). The antibody (anti-clone 22 or anti-IMP.18p proteinantibody) may be produced by any of a number of means known to those ofskill in the art as described herein.

[0184] Immunoassays also often utilize a labeling agent to specificallybind to and label the binding complex formed by the capture agent andthe analyte. The labeling agent may itself be one of the moietiescomprising the antibody/analyte complex. Thus, the labeling agent may bea labeled clone 22 or IMP.18p protein or a labeled anti-clone 22 oranti-IMP.18p protein antibody. Alternatively, the labeling agent may bea third moiety, such as another antibody, that specifically binds to theantibody/clone 22 protein complex.

[0185] In some embodiments, the labeling agent is a second clone 22 orIMP.18p protein antibody bearing a label. Alternatively, the secondclone 22 or IMP.18p protein antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second can bemodified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as enzyme-labeledstreptavidin.

[0186] Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom, et al. (1985) J. Immunol., 135: 2589-2542).

[0187] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 101C to 40°C.

[0188] While the details of the immunoassays of the present inventionmay vary with the particular format employed, the method of detecting aclone 22 or IMP.18p protein in a biological sample generally comprisesthe steps of contacting the biological sample with an antibody whichspecifically reacts, under immunologically reactive conditions, to theclone 22 or IMP.18p protein. The antibody is allowed to bind to theclone 22 or IMP.18p protein under immunologically reactive conditions,and the presence of the bound antibody is detected directly orindirectly.

[0189] A. Non-Competitive Assay Formats

[0190] Immunoassays for detecting clone 22 or IMP.18p proteins of thepresent invention include competitive and noncompetitive formats.Noncompetitive immunoassays are assays in which the amount of capturedanalyte (in this case clone 22 or IMP.18p protein) is directly measured.In one preferred “sandwich” assay, for example, the capture agent(anti-clone 22 or anti-IMP.18p protein antibodies) can be bound directlyto a solid substrate where they are immobilized. These immobilizedantibodies then capture clone 22 or IMP.18p protein present in the testsample. The clone 22 or IMP.18p protein thus immobilized is then boundby a labeling agent, such as a second human clone 22 or IMP.18p proteinantibody bearing a label. Alternatively, the second clone 22 or IMP.18pprotein antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second can be modified with adetectable moiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

[0191] B. Competitive Assay Formats

[0192] In competitive assays, the amount of analyte (clone 22 or IMP.18pprotein) present in the sample is measured indirectly by measuring theamount of an added (exogenous) analyte (clone 22 or IMP.18p protein)displaced (or competed away) from a capture agent (anti clone 22 orIMP.18p protein antibody) by the analyte present in the sample. In onecompetitive assay, a known amount of, in this case, clone 22 or IMP.18pprotein is added to the sample and the sample is then contacted with acapture agent, in this case an antibody that specifically binds clone 22or IMP.18p protein. The amount of clone 22 or IMP.18p protein bound tothe antibody is inversely proportional to the concentration of clone 22or IMP.18p protein present in the sample.

[0193] In some embodiments, the antibody is immobilized on a solidsubstrate. The amount of clone 22 or IMP.18p protein bound to theantibody may be determined either by measuring the amount of clone 22 orIMP.18p protein present in a clone 22 or IMP.18p protein/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed clone 22 or IMP.18p protein. The amount of clone 22 orIMP.18p protein may be detected by providing a labeled clone 22 orIMP.18p protein molecule.

[0194] A hapten inhibition assay is another preferred competitive assay.In this assay a known analyte, in this case clone 22 or IMP.18p proteinis immobilized on a solid substrate. A known amount of anti-clone 22 oranti-IMP.18p protein antibody is added to the sample, and the sample isthen contacted with the immobilized clone 22 or IMP.18p protein. In thiscase, the amount of anti-clone 22 or IMP.18p protein antibody bound tothe immobilized clone 22 or IMP.18p protein is inversely proportional tothe amount of clone 22 or IMP.18p protein present in the sample. Againthe amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

[0195] Immunoassays in the competitive binding format are also used forcrossreactivity determinations to permit one of skill to determine if anovel protein is a homologue, allele, or polymorphic variant of theIMP.18p polypeptide having the sequence set forth as SEQ ID NO:17, thusfalling within the scope of the claimed invention. In this assay, theIMP.18p polypeptide with the sequence set forth as SEQ ID NO:17 isimmobilized to a solid support. Putative IMP.18p polymorphic variantsare added to the assay to compete with immobilized IMP.18p antigen forbinding to a characterized anti-IMP.18p antisera. The ability of theputative IMP.18p polymorphic variants to compete with immobilizedIMP.18p antigen for binding to the anti-IMP.18p antisera is compared tothe ability of IMP.18p of SEQ ID NO:17, or immunogenic fragmentsthereof, to compete with immobilized antigen for binding to theantisera. The percent crossreactivity for the above proteins iscalculated, using standard calculations.

[0196] To prepare the antisera for use in this competitive bindingimmunoassay, all IMP cross-reacting antibodies are first removed byimmuno-absorption with known IMP polypeptides. Specifically, antiseraare immunosorbed with the human IMP (huIMP) defined by McAllister (1992)Biochem J. 284:749-754. GenBank Accession #P29218; bovine IMP defined byYork (1990) Proc. Natl. Acad. Sci. USA 87:9548-9552, GenBank Accession#P21327; and, rat IMP as defined by Parthasarathy (1997) Gene 191:81-87.GenBank Accession #U84038. Antisera with less than 10% crossreactivitywith non-IMP.18p/SEQ ID NO:17 polypeptides are selected and pooled(i.e., 90% of the antisera is non-cross reactive, thus specific). Thus,the anti-IMP.18p antibodies and antisera of the invention have less than10% cross-reactivity to (e.g., as they are immunosorbed against)previously characterized anti-IMP polypeptides, as discussed above. Theimmunoabsorbed antisera are used in a competitive binding immunoassay,as described below, to analyze whether an uncharacterized protein is anIMP.18p protein within the scope of the claimed invention.

[0197] In this competitive binding immunoassay, the IMP.18p protein ofSEQ ID NO:17 competes with a second, putative IMP.18p polymorphicvariant in an antibody binding reaction. The known and uncharacterizedIMP.18p polypeptides are competitively reacted with antisera developedagainst and specifically reactive with the IMP.18p of SEQ ID NO:17(antisera immunosorbed to ensure no cross-reactivity with previouslycharacterized IMPs, as described above). The two polypeptides are eachassayed at a wide range of concentrations. The amount of eachpolypeptide required to inhibit 50% of the binding of the anti-IMP.18p(SEQ ID NO:17) antisera to immobilized IMP.18p (SEQ ID NO:17)polypeptide is determined. If the amount of the second (uncharacterized)protein required is less than 10 times the amount of the characterizedimmunogen (IMP.18p/SEQ ID NO:17) that is required, then the secondprotein is said to specifically bind to an antibody generated to thecharacterized (IMP.18p/SEQ ID NO:17) immunogen.

[0198] Immunoassays in the competitive binding format can be used forcrossreactivity determinations to permit one of skill to determine if anovel anti-IMP.18p antibody or antisera is sufficiently related to theanti-IMP.18p polypeptide of the invention with the sequence set forth asSEQ ID NO:17 so as to fall under (within the scope of) the claims ofthis invention. For example, the IMP.18p/SEQ ID NO:17 polypeptide isimmobilized to a solid support. Test antibodies are added to the assayto compete with the binding of the known anti-IMP18.p/SEQ ID NO:17antisera to the immobilized antigen (IMP.18p/SEQ ID NO:17). The abilityof the test antisera to compete with the binding of the known antiserato the immobilized IMP.18p is compared. The percent crossreactivity forthe above antibodies is calculated, using standard calculations.

[0199] C. Other Assay Formats

[0200] In other embodiments. Western blot (immunoblot) analysis is usedto detect and quantify the presence of clone 22 or IMP.18p protein inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind clone22 or IMP.18p protein. The anti-clone 22 or anti-IMP.18p proteinantibodies specifically bind to clone 22 or IMP.18p protein,respectively, on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-clone 22 or anti-IMP.18p protein.

[0201] Assaying for Activity and Modulators of IMP.18p Myo-InositolMonophosphatase

[0202] The invention also provides for means to assay the activity ofthe novel IMP18.p myo-inositol monophosphatase enzyme. Using suchassays, one embodiment provides for a method of determining whether atest compound is a modulator, such as an inhibitor/antagonist oragonist, of IMP.18p myo-inositol monophosphatase activity. The methodinvolves contacting an active IMP.18p with a putative modulator testcompound and measuring the activity of the IMP.18p. A change in theactivity of the IMP.18p in the presence of the test compound is anindicator of whether the test compound is an antagonist oragonist/activator of IMP.18p. A variety of myo-inositol monophosphataseactivity assays are known in the art which can be adapted by the skilledartisan to be used using the novel IMP.18p in the methods of theinvention. Illustrative examples of such assays are set forth below.

[0203] Myo-inositol monophosphatases are major enzymes controlling theinositol intracellular signaling pathway. Numerous diacylglycerol andcalcium-mobilizing enzymes are associated with this pathway, includingserotonergic, muscarinic, adrenergic, metabotropic, histaminergic,cholecystokinin, tachykinin, bombesin, neurotensin and bradykininreceptors, to name a few examples. Activation of these receptorsactivates GTP binding proteins, which results in the phospholipase Chydrolysis of inositol-phospholipid. This reaction releases twointracellular messengers: myo-inositol 1,4,5-triphosphate (IP3) anddiacylglycerol (DAG). IP3 releases intracellular calcium stores, whichin turn activates a variety of second signals, triggering numerousphysiologic effects, for example, ion channel activation. Levels of IP3are controlled by sequential dephosphorylation, the last step generatingthe products inositol and phosphate from the substrate myo-inositolmonophosphate (myo-inositol 1-phosphate) by the enzyme myo-inositolmonophosphatase. Thus, the activity of myo-inositol monophosphates canbe monitored in vitro or in vivo by measuring the loss or accumulationof a substrate or a product, respectively, over time.

[0204] Monitoring the activity and assessment of potential modulators ofthe novel IMP.18p of the invention can be accomplished in vitro bymeasuring the accumulation of either myo-inositol monophosphataseproduct in the form of radiolabeled inositol (e.g., ¹⁴C-inositol or³H-inositol) or inorganic phosphate (Pi) (e.g., in a colorimetric assayor as ³²Pi). For example, a Pi-release assay based on colorimetric meansto measure changes in Pi concentration over time can be carried out asdescribed by Ragan (1988) Biochem. J. 249:143-148. or, by Vadnal (1995)Neuropsychopharmacol. 12:277-285.

[0205] As in Vadnal (1995) supra, the reaction mixture can consist of0.05 ml of 120 mM Tris-HCl, pH 7.8; 0.05 ml of 18 mM or 3 mM magnesiumchloride; 0.05 ml of 4.2 mM D-myo-inositol 1-phosphate, 0.125 ml wateralone or with positive controls or putative modulator test compounds orcompositions. Known myo-inositol monophosphatase inhibitors(antagonists), such as lithium, carbamazepine and/or valproic acid, invarying amounts can be used as controls. A 0.025 ml solution ofmyo-inositol monophosphatase (e.g., IMP.18p, or another myo-inositolmonophosphatase as a positive control) is added and the reaction mixtureis incubated at 37° C. for about 15 minutes to an hour. The reaction isstopped by the addition of 0.05 nl of 20% trichloroacetic acid (TCA).The suspension is centrifuged and 0.10 ml of supernatant is used toestimate the liberated Pi using the malachite green reagent method, as,for example, described by Eisenberg (1987) Methods Enzymol. 141:127-143.Protein is assayed using the method of Lowry (1951) J. Biol. Chem.193:265-275. Assays are usually run in triplicate. Alternatively, as inRagan (1988) supra, the reaction mixture can be in a final volume of0.300 ml containing 0.1 mM substrate, 250 mM potassium chloride, 50 mMTris HCl, pH 8.0, and 3 mM magnesium chloride for period of time from 15minutes to one hour. Released Pi can be measured calorimetrically usingthe method of Itaya (1966) Clin. Chem. Acta 14:361-366 (see also Kodama(1986) “The initial phosphate burst in ATP hydrolysis by myosin andsubfragment-1 as studied by a modified malachite green method fordetermination of inorganic phosphate,” J. Biochem. (Tokyo)99:1465-1472). The specific activity of myo-inositol monophosphatase isexpressed as nanomoles of phosphate liberated per minute (mU) permilligram protein.

[0206] Kinetic activity and assessment of potential modulators of theIMP.18p of the invention can also be accomplished in vivo by measuringaccumulation of the substrate myo-inositol monophosphate (myo-inositol1-phosphate) using, for example, assays described by Atack (1993) J. ofNeurochem. 60:652-658; or, Ragan (1988) supra. Radiolabeled inositolmonophosphate accumulation can be measured in tissue culture cellsexpressing IMP.18p in the presence of putative myo-inositolmonophosphatase antagonists, for example, as described by Atack (1993)supra. The tissue culture cells can be genetically manipulated, asdescribed above, to express the IMP.18p of the invention, or fragmentsor variations thereof. For example, as described above, CHO cells can bemanipulated to express very large amounts of exogenous protein.Specifically, to assess the effect of a putative antagonist or agoniston myo-inositol monophosphatase in vivo, CHO cells are first pre-labeledwith ³H-inositol. Prelabeling involves growing cells to confluence fortwo days in medium containing radiolabeled inositol (e.g., ¹⁴C-inositolor ³H-inositol). If using ³H-inositol, 0.5 uCi/ml 80 Ci/mmol (AmershamInternational) is used. On the day of the experiment, cells areharvested in Krebs-Henseleit buffer at 2×10⁶ cells/ml containing 0.5uCi/ml 3H-inositol. Aliquots of the harvested cells are incubated forone hour at 37° C. in a shaking water bath in the presence of 10 ul ofvarious concentrations of known enzyme inhibitors and testcompounds—putative enzyme modulators. Assays are terminated by additionof 300 ul of 1.0 M TCA and centrifuged. 500 ul of supernatant is washedwith water-saturated diethyl ether. The pH is adjusted to about 7.0using 1 M Tris. The supernatants are then applied to Dowex columns.Columns are washed four times with 5 ml of water to elute free³H-inositol; then washed three times with 5 ml of 25 mM ammonium formateto elute beta-glycerophosphates. ³H-inositol 1-monophosphate iscollected by washing the column with 10 ml of 200 mM ammonium phosphateand counted on a scintillation counter. Alternatively, ¹⁴C-inositol canbe used, as described by Ragan (1988) supra. Inhibition of themyo-inositol monophosphatase will result in increased levels of thesubstrate myo-inositol monophosphate (myo-inositol 1-phosphate), whileactivation of the enzyme will result in decreased levels of substrateand increased levels of product (inositol and inorganic phosphate).

[0207] Using these assays and variations thereof, the kinetics of theIMP.18p enzyme with and without test modulators (e.g., competitive ornon-competitive antagonists) can be analyzed using known methods (e.g.,Lineweaver-Burke plots, as used, for example by Lee (1996) Xenobiotica26: 831-838); for discussion on enzyme kinetic analysis generally see,for example, Suarez (1997) Proc. Natl. Acad. Sci. USA 94:7065-7069;Northrop (1997) Bioorg. Med. Chem. 5:641-644); Sterrer (1997) J. Recept.Signal Transduct. Res. 17:511-520).

[0208] High-Throughput Screening of Candidate IMP.18p Modulators

[0209] Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity (in this case, e.g., anantagonist or agonist of IMP.18p), creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

[0210] In one preferred embodiment, high throughput screening methodsinvolve providing a library containing a large number of potentialtherapeutic or diagnostic compounds (candidate compounds). Such“combinatorial chemical libraries” are then screened in one or moreassays, some of which are described above, to identify those librarymembers (particular chemical species or subclasses) that display thedesired characteristic activity (e.g., modulation of the activity ofIMP.18p). The compounds thus identified can serve as conventional “leadcompounds” or can themselves be used as potential or actualtherapeutics. See also, van Breemen (1997) Anal Chem 69:2159-2164; Lam(1997) Anticancer Drug Des 12:145-167 (1997).

[0211] a. Combinatorial Chemical Libraries

[0212] Recently, attention has focused on the use of combinatorialchemical libraries to assist in the generation of new chemical compoundleads. A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library, such asa polypeptide library is formed by combining a set of chemical buildingblocks called amino acids in every possible way for a given compoundlength (i.e., the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

[0213] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res. 37:487-493, Houghton et al. (1991) Nature, 354: 84-88).

[0214] Peptide synthesis is by no means the only approach envisioned andintended for use with the present invention. Other chemistries forgenerating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (PCT PublicationNo WO 91/19735, Dec. 26, 1991), encoded peptides (PCT Publication WO93/20242, Oct. 14, 1993), random bio-oligomers (PCT Publication WO92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No. 5,288,514),diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs etal., (1993) Proc. Natl. Acad. Sci. USA 90: 6909-6913), vinylogouspolypeptides (Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568),nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding(Hirschmann et al., (1992) J. Amer. Chem. Soc. 114: 9217-9218),analogous organic syntheses of small compound libraries (Chen et al.(1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al.,(1993) Science 261:1303), and/or peptidyl phosphonates (Campbell et al.,(1994) J. Org. Chem. 59: 658). See, generally, Gordon et al., (1994) J.Med. Chem. 37:1385, nucleic acid libraries, peptide nucleic acidlibraries (see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see,e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines 5,288,514, and thelike).

[0215] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech.Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).

[0216] A number of well known robotic systems have also been developedfor solution phase chemistries. These systems include automatedworkstations like the automated synthesis apparatus developed by TakedaChemical Industries, LTD. (Osaka, Japan) and many robotic systemsutilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.;Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manualsynthetic operations performed by a chemist. Any of the above devicesare suitable for use with the present invention. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art. In addition, numerous combinatorial libraries arethemselves commercially available (see, e.g., ComGenex, Princeton. N.J.,Asinex, Moscow, Ru, Tripos, Inc. St. Louis, Mo., ChemStar, Ltd, Moscow,RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,etc.).

[0217] b. High Throughput Assays of Chemical Libraries

[0218] Any of the assays for compounds inhibiting the virulencedescribed herein are amenable to high throughput screening. As describedabove, having identified the nucleic acid associated with virulence,likely drug candidates either inhibit expression of the gene product, orinhibit the activity of the expressed protein. Preferred assays thusdetect inhibition of transcription (i.e., inhibition of mRNA production)by the test compound(s), inhibition of protein expression by the testcompound(s), or binding to the gene (e.g., gDNA, or cDNA) or geneproduct (e.g., mRNA or expressed protein) by the test compound(s).Alternatively, the assay can detect inhibition of the characteristicactivity of the gene product or inhibition of or binding to a receptoror other transduction molecule that interacts with the gene product.

[0219] High throughput assays for the presence, absence, orquantification of particular nucleic acids or protein products are wellknown to those of skill in the art. Similarly, binding assays aresimilarly well known. Thus, for example, U.S. Pat. No. 5,559,410discloses high throughput screening methods for proteins, U.S. Pat. No.5,585,639 discloses high throughput screening methods for nucleic acidbinding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541.061disclose high throughput methods of screening for ligand/antibodybinding.

[0220] In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high thruput and rapid start up as well as a high degreeof flexibility and customization. The manufacturers of such systemsprovide detailed protocols the various high throughput. Thus, forexample. Zymark Corp. provides technical bulletins describing screeningsystems for detecting the modulation of gene transcription, ligandbinding, and the like.

[0221] Rational Drug Design

[0222] Potential modulators of enzyme activity can also be investigatedutilizing “rational drug design” approaches. This involves an integratedset of methodologies that include structural analysis of targetmolecules, synthetic chemistries, and advanced computational tools. Whenused to design modulators, such as antagonists/inhibitors of proteintargets, such as IMP.18p polypeptides, the objective of rational drugdesign is to understand a molecule's three-dimensional shape andchemistry. Rational drug design is aided by X-ray crystallographic dataor NMR data, which can now be determined for the IMP.18p polypeptide inaccordance with the methods and using the reagents provided by theinvention. Calculations on electrostatics, hydrophobicities and solventaccessibility is also helpful. See, for example, Coldren (1997) Proc.Natl. Acad. Sci. USA 94:6635-6640.

[0223] Inhibitory Natural Compounds as Modulators of IMP.18p Activity

[0224] In addition, a large number of potentially usefulactivity-modifying compounds can be screened in extracts from naturalproducts as a source material. Sources of such extracts can be from alarge number of species of fungi, actinomyces, algae, insects, protozoa,plants, and bacteria. Those extracts showing inhibitory activity canthen be analyzed to isolate the active molecule. See for example, Turner(1996) J Ethnopharmacol 51(1-3):39-43; Suh (1995) Anticancer Res15:233-239.

[0225] Inhibitory Oligonucleotides

[0226] One particularly useful set of inhibitors provided by the presentinvention includes oligonucleotides which are able to either bind mRNAencoding IMP.18p or clone 22 polypeptides or to their correspondinggenes. In either case, these oligos prevent or inhibit the production offunctional protein.

[0227] Another useful class of inhibitors includes oligonucleotideswhich cause inactivation or cleavage of IMP.18p or clone 22 mRNA. Thatis, the oligonucleotide is chemically modified or has enzyme activitywhich causes such cleavage, such as ribozymes. As noted above, one mayscreen a pool of many different such oligonucleotides for those with thedesired activity.

[0228] Another useful class of inhibitors includes oligonucleotideswhich bind polypeptides. Double- or single-stranded DNA orsingle-stranded RNA molecules that bind to specific polypeptides targetsare called “aptamers.” The specific oligonucleotide-polypeptideassociation may be mediated by electrostatic interactions. For example,aptamers specifically bind to anion-binding exosites on thromoin, whichphysiologically binds to the polyanionic heparin (Bock (1992) Nature355:564-566). Because the present invention provides proteins inpurified form in large quantities, those of skill in the art can readilyscreen for IMP.18p-binding aptamers using the methods of the invention.

[0229] Antisense Oligonucleotides

[0230] IMP.18p or clone 22 activity can be inhibited by targeting theirrespective mRNA with antisense oligonucleotides capable of binding themRNA. In some situations, naturally occurring nucleic acids used asantisense oligonucleotides may need to be relatively long (18 to 40nucleotides) and present at high concentrations. AR wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphorodithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

[0231] As noted above, combinatorial chemistry methodology can be usedto create vast numbers of oligonucleotides that can be rapidly screenedfor specific oligonucleotides that have appropriate binding affinitiesand specificities toward any target, such as the IMP.18p of theinvention, can be utilized (for general background information Gold(1995) J. of Biol. Chem. 270:13581-13584).

[0232] Inhibitory Ribozymes

[0233] Ribozymes act by binding to a target RNA through the target RNAbinding portion of a ribozyme which is held in close proximity to anenzymatic portion of the RNA that cleaves the target RNA. Thus, theribozyme recognizes and binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

[0234] In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

[0235] The enzymatic ribozyme RNA molecule has complementarity to thetarget, such as the mRNA encoding IMP.18p. The enzymatic ribozyme RNAmolecule is able to cleave RNA and thereby inactivate a target RNAmolecule. The complementarity functions to allow sufficienthybridization of the enzymatic ribozyme RNA molecule to the target RNAfor cleavage to occur. One hundred percent complementarity is preferred,but complementarity as low as 50-75% may also be employed. The presentinvention provides ribozymes targeting any portion of the coding regionfor an IMP.18p or clone 22 gene that cleaves their corresponding mRNA ina manner that will inhibit the translation of the mRNA and thus reduceenzymatic activity. In addition, the invention provides ribozymestargeting the nascent RNA transcript of the IMP.18p or clone 22 gene toreduce activity.

[0236] The enzymatic ribozyme RNA molecule can be formed in a hammerheadmotif, but may also be formed in the motif of a hairpin, hepatitis deltavirus, group I intron or RNaseP-like RNA (in association with an RNAguide sequence ). Examples of such hammerhead motifs are described byRossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifsby Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849: and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

[0237] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXAMPLES

[0238] The following examples are offered to illustrate, but not tolimit the claimed invention.

Example 1 Chromosome 18-Specific Cosmid Clones Used for cDNA Selection

[0239] A human chromosome 18-specific cosmid library, LL18NC02, wasprovided by the Human Genome Center at the Lawrence LivermoreLaboratory. The source of the chromosomes was a human/hamster hybridcell line X11-4A (Chang et al., Genomics, 17:393-402, 1993; Trask etal., Somat Cell Mol Gene, 17:117-136, 1991) retaining a single copy ofchromosome 18 as its sole human material. The chromosomal DNA waspartially digested with MboI, dephosphorylated, then ligated into theBamHI site of the cosmid vector Lawrist 16 (Little, PFR (1987): Choiceand use of cosmid vectors. In Glover DM (ed): “Gene Cloning” Vol. 3, IRLPress: Oxford, pp 19-42). The resulting arrayed library contained 14596-well microtiter plates. A human genomic DNA probe hybridized to 84%of the clones in the library, 10% were positive with a rodent probe andthe remaining 6% were non-recombinants since they failed to hybridizewith either probe. The chromosome 18 cosmid library represents 467 Mb[13.920 clones×94%×40 kb (assumed average size of cosmid insert)] inchromosomal coverage. Ten pools of the library were prepared bycombining the contents of all wells from plates 1-10 (pool 1), 11-25(pool 2), 26-40 (pool 3), etc. Cultures of the cosmid pools were grownin LB/kanamycin and the DNA isolated using the Qiagen plasmid kit(Qiagen). The DNA was biotinylated for 20 minutes using the Bio-Nick kit(GIBCO-BRL). The unincorporated nucleotides were excluded by ethanolprecipitation.

Example 2 Preparation of Primary cDNA

[0240] Total RNA was extracted from five regions of postmortem humanbrain (caudate, putamen, hippocampus, amygdala, frontal cortex) and fromhuman placenta by acid-guanidine, phenol/chloroform method (Chomzynskiand Sacchi, Anal Biochem, 162:156-159, 1987). Poly(A)+ RNA was preparedusing oligo(dT)-paramagnetic beads (Dynal), and double stranded cDNA wassynthesized with random priming using the Invitrogen Copy kit. The cDNAwas subdivided into eight pools, each containing 1 μg of brain-derivedcDNA and 0.8 μg of placental cDNA. A batch of total human brain poly(A)+RNA was purchased from Clontech (in order to represent regions of thebrain not included above), and 4 μg of double stranded cDNA was preparedas above. Each cDNA pool was ligated to an adaptor consisting ofcomplementary oligonucleotides 1 and 2 (Lovett, Proc Natl Acad Sci USA,88:9628-9632, 1994). Since the brain tissues obtained were frozenfollowing a postmortem delay, placental cDNA was added in the selectionto retain transcripts (common to both brain and placenta) that mighthave been labile during this delay.

Example 3 Direct cDNA Selection

[0241] Direct cDNA selection was performed using the magnetic beadcapture technique described previously (Lovett et al. Proc Natl Acad SciUSA, 88:9628-9632, 1991; Lovett, “Current Protocols in Human Genetics”Vol. 1, John Wiley & Sons Inc: New York, pp 6.3.1-6.3.15, 1994) to aCot_(1/2) of 100, with some modifications. Briefly, repeats were blockedby mixing the starting cDNA pool with a mixture of low molecular weightCot-1 DNA (2 μg per hybridization, GIBCO-BRL), high molecular weightCot-1 DNA (20 ng per hybridization, GIBCO-BRL) and linearized cosmidvector DNA (30 ng per hybridization). The first round of selection wasperformed by hybridization of cDNA pools (1.8-2 μg each) andbiotinylated cosmid pools (120 ng each). A second round of selection wasconducted using 2 μg of amplified primary-selected cDNA and 120 ng ofeach biotinylated pool of cosmids. The PCR reactions for the primary-and secondary-selected cDNAs were performed using Expand Long TemplatePCR System (Boehringer Mannheim) with an initial denaturation at 94ECfor 3 min, followed by 10 cycles of amplification at 94EC for 10 sec.60EC for 30 sec and 68EC for 3 min, and 25 cycles using the samedenaturation and annealing conditions, and an auto-extended elongationtime of an additional 15 sec after every cycle.

Example 4 Hybridization of High Density Filters of a Normalized InfantBrain cDNA Library

[0242] Approximately 40,000 clones from a normalized infant brainlibrary constructed by Soares et al. (1994), Proc Natl Acad Sci USA,91:9228-9232, were previously arrayed at the Lawrence LivermoreLaboratory into 408 96-well microtiter plates. We re-arrayed the libraryinto 102 384-well microtiter plates and high density filters wereproduced (service done by Research Genetics, Inc). One 22×22 cm filtercontained 36,864 clones and the remaining 2,304 clones were spotted onanother filter.

[0243] Each pool of amplified secondary-selected cDNA was labeled withgamma-³²P-dCTP by random primer labeling (Boehringer Mannheim kit). Oneset of hybridizations of the high density filters was done using amixture of all the pools of labeled secondary selected cDNA, after apreblocking procedure using total human placental DNA, low molecularweight Cot-1, and linearized cosmid vector. Hybridization was done using2×10⁶ cpm of preblocked cDNA per ml of Rapid-hyb buffer (Amersham) at65EC for 2 hrs following a prehybridization of 1 hr. The final wash wasin 0.1×SSC, 0.1% SDS at 60EC. Using the same conditions, a replicafilter was hybridized with-5×10¹⁰ ⁵ cpm per ml of ³²P-labeled humanplacental DNA.

[0244] Another set of hybridizations was performed using a mixture oftwo pools of secondary-selected cDNA. The hybridization pattern yieldedby the secondary selected cDNAs was compared with that produced by humanplacental DNA. The clones corresponding to positive spots common to bothfilters were not picked due to the possibility that the signals werefrom repeat hybridization. In addition, the hybridization patternobtained with the cDNA subpools were compared with that produced using acombination of all secondary selected cDNA pools. All high and mediumintensity clones were chosen, and clones that gave low intensity signalsbut were common to two or more filters were also picked. The insertsizes were determined using the colony PCR method described previously(Yoshikawa et al. Biochim Biophys Acta. 1264:63-71, 1995).

Example 5 Sequence Database Comparisons and Primer Design

[0245] The microtiter plate addresses of the positive clones chosen forfurther analysis were determined, and this allowed us to search the ESTdatabase (dbEST) (Boguski et al., Nature Gene. 4:332-333, 1993; URL:http//ncbi.nlm.nih.gov/Schuler/Onigene/Chr18.html, searched on Mar. 18,1996) permitting the retrieval of the IMAGE cDNA ID number andcorresponding Genome Database (GDB) account number. Approximately 40% ofthe cDNA clones contained a short 3′ and/or 5′ end STSs that weredeposited by the sequencing collaboration of Washington University andMerck & Co. For these available sequences, primers were designed usingthe program PRIMER v2.2 (Resnick and Stein, Primer, v 2.2. The WhiteheadInstitute, Cambridge, Mass., 1995; URL: http://www-genome.wi.mil.edu),which had a Tm for primers set at between 52° C. and 55° C. (see Table2, below).

Example 6 Mapping of cDNA Clones by PCR on Chromosome 18 Somatic CellHybrids

[0246] Genomic DNA was extracted from a panel of 20 somatic cellhybrids, one of which included the entire human chromosome 18 and therest containing various segments of the chromosome (Overhauser et al.Cytogenet Cell Gene, 71:106-117, 1995). A diagram of the hybrids used inthis study is shown in FIG. 1. Human genomic DNA and hamster genomic DNAwere used as reference controls. Using this panel of chromosomal andgenomic DNA as template and primer pairs derived from each clone mappingby PCR was conducted. If the initial primer pair failed to amplify,another pair was designed, or one of the primers in the original pairwas modified.

[0247] PCR was performed using the Perkin Elmer Cetus GeneAmp System9600. Amplification was done in a 20 μl reaction containing either 80 ng(somatic cell hybrid) or 30 ng (human or hamster genomic DNA) templateDNA, 5 μM of each primer, 200 μM of each dNTP and 0.75 unit of AmpliTaq(Perkin Elmer Cetus) in a standard PCR I buffer TABLE 2 TABLE 2: Primersused for PCR mapping. Clone Primer Sequence Product Number Forward SEQID NO: Reverse SEQ ID NO: Size (bp) 1 5′-AGGAGTGGTGTACATTTCT-3′ 1105′-ACCTGCAACACATTAGAAAC-3′ 111 134 2 GGTTTCTTCAAAATTTTATTAACAA 112TCCTCCACTCATCTGTTTCT 113 175 3 CCTGACCTGATCAAGTTTA 114GGTAAAGGAACAAGCTGC 115 125 4* TGATCACAOAGTCAGCACTGT 116GGGCAGAAGTTTCCAATTACC 117 131 5 TATTGAGACCTAAGTCAGCATCC 118GACAGAAAGCAGGTTAGAGGT 119 192 6 GAAACTTTACATCAGGTGTCTC 120ATGGACTAGGAGTTTAAGC 121 283 7 GGAACAGTGTACACTTTCC 122TATATAGCCTCGATGATGAGAG 123 185 8 CATGAGAGGAAGAGGTCTTTAT 124GGGTAATGTCTTAGTCGAG 125 275 9 TCAGTAGAAACTCAAGCTGCTTC 126CTCCCTCTCAGTGTGAGGCT 127 230 10 CCTGACCTGATCAAGTTTAA 128TGTACACCACTCCTCATGT 129 179 11 CGACGACTCATACAACATATC 130GGTTACAGCTGAAGTGTAT 131 177 12 TATTCAGGAACAGTGTACAC 132TCGATGATGAGAGGGTTAC 133 174 13 GAACACTTATCTCCTTCTTCAG 134TCCACTCCTTTCACCTCTTCT 135 243 14 AGACAAGAGCAAAACACAAC 136CTCTTTGCAGTTCAGTCTA 137 169 15 AGGTGAACCATTTGACTGGTTT 138GCTTGTGTGTGGCTGTCCTT 139 148 16 GGCTAAACTTACAGTATGTAAGGAG 140CTGTAAGGACAGACTACTCA 141 152 17 CCAGGAGGTTTCAGCGGT 142CGCAAAGCCATGATAAACCG 143 115 18^(A) TCAGGAACAGTGTACACTTTC 144TGTGGGCTTAATACCATGTCT 145 207 19 GGAATCTCTGTACTTGCT 146GTGACACATTACAAAGCCA 147 154 20 TCAGTAGAAACTCAAGCTGC 148CCTCTTCCTCTTTAAGTGT 149 101 21 TCACTTCAGAATCACTACTC 150ACCCATCCTATATGAAAAGC 151 228 22 TACAAAAGAGGACAAAGCAC 30GGTGCCTGTATATAAGTTGA 32 157 23 GGGATCATACTAAAGAGAAG 152GGATAAACAGAGAGCTTGAT 153 193 24 CTACAGAATAGAATACATGGCG 34GAGCTCTGAACTGTATTCAGA 36 224 25 GTCAGTTACTCTATTTGCTGTG 154AACCTGTGCTGTAAAGTTCA 155 233 26 CTTAAGAGGAAGAGGCCAT 156CTCTCCCTCTCAGTGTGAG 157 145 27 ACAATTAGGCATTGTTGATGG 158CAGTTCTTGCACATACAAGACA 159 112 28 ACCTTTGGCAAGGGGTATGA 160TGTGAAGGCTGGGAAACACT 161 207 29 TCTCAGCTTACTCAACCT 38GATGAGGTGGAACAATCAC 40 138 30 AACACTCAGCTCTGTAGAA 162CGAGTCATCAATAGGACAA 163 212 31 GGTCTGTACAGTGTAATAAACC 42CTACTGCAAAATGTGTCCTGTC 44 124 32 GAGCCAAGTGGAACTCTTGAA 164GTCAGGAAAGAGGTTGTGAGC 165 156 33 ACACATATGTACACAGGAAC 166TGTGTACAGCGAGTGAATTA 167 103 34 TTGTTCACACACAATCTAGG 168ACTAGCATATCTGAATTCCCA 169 159 35 CTACAGAATAGAATACATGGCG 170TTGAAACCAGACCCTGTAGT 171 166 36 CATTTAGTCCAGAGGCTCTT 172TCCTCGAAGAGGTTGCAGC 173 161 37 CACATTAGCCAGTCTGATAAAG 46AAGTTACACACAGTAGCTGA 48 107 38 CATTCAGCACACATAGAGTCTA 174CCCTGTCCCTTGTATATGTA 175 189 39 AGTGTATCTACAACCTCAACTGTC 176GTAAAGGCCCAATCAATGCACT 177 109 40 GOCAGATTCACAATTGATAG 178CTGAAGGCACTTTATGTAC 179 139 41 CTGGAGCAGGTTAGATACACC 180CTTCCCTCTTAACCTTTAGTGC 181 143 42 GTGTCTTGTATGTGCAAGAAC 182GACTGGGTATCCTAGCTTAC 183 157 43^(A) TTAGTCAGACCCATTCAGTC 184CCAGACTGCTTTATGTTAG 185 103 44^(A) GTGTCTTGTATGTGCAAGAAC 186CCTAGCCTTACTGTTTTAAC 187 146 45* ACGATGCGATCCTGGAAG 188CTGGCTTGAGTTTGTCTG 189 113 46† CCTTTCTGTGTGAAGATCAC 190AAGAAAGTCCCAAGGGTGGA 191 123 47† GGAATGAGGGTTAGAGTCC 192AGTGCTTCTGTAGCTCTT 193 114 48 TGAGGGTGTGAACCACTCTG 194GAATCCTGGTGTGCCCAAGT 195 137

[0248] (Perkin Elmer Cetus). “Touchdown” PCR was done as follows: 30 secat 94EC, 30 sec at (T+11−n)EC (T is listed in Table 2 and n is cyclenumber), 1 min at 72EC for the first 10 cycles, and 30 sec at 94EC, 30sec at TEC, 1 min at 72EC in the subsequent 25 cycles. The PCR productswere separated on 3% Nusieve:Seakem agarose gels.

Example 7 Radiation Hybrid Mapping

[0249] The Stanford G3 radiation hybrid panel (Cox et al., Science,250:245-250, 1990) (#RH01, available at Research Genetics, Inc.) wasutilized to fine map the unique chromosome 18-specific brain cDNAs. Thispanel had a 500 kb resolution and an average of 26 kb per centiRay (cR),based on data available for chromosome 4 on 452 informative markers(http://shgc.stanford.edu/RHMap.html).

[0250] For radiation hybrid mapping, 40 ng of DNA from each of the 83radiation hybrid cell lines were used as template, and PCR was performedwith primers specific for a given cDNA clone (Table 3).

[0251] PCR was done in a 10 μl volume, and conditions were identical tothose previously described for mapping with the chromosome 18 regionalpanel of somatic cell hybrids. Fifteen ng of human genomic DNA was usedas positive control. The size of a PCR product, amplified from eachradiation hybrid cell line, and a given pair of primers was determinedby electrophoresis on a 3% Nusieve:Seakem agarose gel. For a givenprimer pair, the raw data indicating the presence or absence of anamplified product in each of the 83 radiation hybrid cell lines wassubmitted to the Stanford radiation hybrid e-mail server(http://shgc.stanford.edu/rhserver/intro.html). If linkage to referencemarkers was found, the mapping data transmitted from Stanford included alist of linked markers (STSs), lod scores and distances in cR₍₈₀₀₀. Alod score above 6 was used for assigning the unique clones to theStanford framework map with a 95% confidence level.

Example 8 cDNA Selection and Isolation of cDNA Clones from an InfantBrain Library

[0252] To isolate brain-expressed transcripts that map specifically tochromosome 18, we performed direct cDNA selection with pools ofchromosome 18 biotinylated cosmid clones and primary cDNAs derived fromhuman brain and placenta. After two cycles of selection, the secondaryselected cDNA was PCR amplified, and this was found to have an averagesize of about 400 bp. Longer cognate cDNA clones were isolated by usinglabeled amplified pools of secondary selected cDNAs to probe highdensity filters of an arrayed, normalized infant brain library (Soareset al., Proc Natl Acad Sci USA, 91:9228-9232, 1994). This strategyyielded a total of 174 positive cDNA clones. Analysis of the dbESTdatabase revealed that less than half of these clones had availablesequences of a few hundred bp on the 3′ and/or the 5′ ends.

[0253] Initially, we focused our analysis on clones that had thesepartial sequences to facilitate rapid chromosomal localization by PCR.The availability of these sequences also permitted comparison withsequences in the databases for homology to known genes, and evaluationof possible redundancies between the selected transcripts.

Example 9 Chromosomal Localization and Regional Mapping of Chromosome18-Specific cDNAs

[0254] To determine the chromosomal location of the positive cDNA cloneswe designed PCR primers from the 3′ end sequence, whenever possible.Since the infant brain cDNA library was constructed by oligo (dT)priming and directional cloning this would most likely correspond to the3′ untranslated region (UTR), which is usually unique and uninterruptedby introns (Sikela and Auffray, Nature Gene 3:189-191, 1993). Primerswere developed to produce PCR products of less than 300 bp. Our analysisindicated that 83% of 3′ end-derived primer pairs and 74% of 5′end-derived primer pairs amplified a PCR product with the expected size.

[0255] In the initial step of the clone-based physical mapping, a panelof template DNAs was used for PCR amplification. These included: humanplacental DNA, somatic cell hybrid DNAs for the entire human chromosome18 (HHW 324, FIG. 1) as well as segments (JH 353 and JH357, FIG. 2) ofhuman chromosome 18, and hamster DNA. In addition, a number of somaticcell hybrid DNA isolates derived from other chromosomes were used asnegative controls. After establishing that the cDNA was of human originand was specifically localized to chromosome 18, mapping intosubchromosomal regions was performed by PCR on a series of DNAs derivedfrom somatic cell hybrids that subdivide the cytogenetic bins (FIG. 1).

[0256] We found that the use of primers derived from 48 cDNA clonessuccessfully chromosome into amplified unique bands of the expectedsize, specifically on chromosome 18 somatic cell hybrid DNA (Table 2).Further analysis using the same primer pairs (Table 2) revealed thateach of these 48 clones mapped to a specific chromosome 18 cytogeneticbin (Table 3 and FIG. 1), therefore, confirming our initial data on thechromosomal assignment. The remaining clones mapped either ambiguouslyor elsewhere in the genome.

[0257] Interestingly, most of 48 brain transcripts appeared to clusterwithin discrete cytogenetic regions on chromosome 18; bins A and B, inthe short arm and bins M and S, in the long arm (Table 3 and FIG. 1).

Example 10 Sequence Homology Comparisons to Identify Unique Chromosome18-Specific Transcripts

[0258] To determine the identity and uniqueness of each of the 48chromosome 18-specific transcripts, a homology search against sequencedatabases was conducted. By comparison using a BLASTN similarity searchwith GenBank (Altschul, J. Mol Biol, 215:403-410, 1990) and a Level Isequence EST homology search of The Institute for Genome Research (TIGR)database (Adans et al., Nature, 377 (Suppl.):3-174, 1995), we found thatof the 48 chromosome 18-specific cDNAs, 11 were highly homologous(defined as >89% homology over >100 bp) to segments of five previouslyknown genes (see Table 4, below).

[0259] Myelin basic protein (MBP, Kamholz et al., Proc Natl Acad SciUSA, 83:4962-6, 1986), the 63 kDa protein kinase related to ERK3 (HS63KDAP, Li et al., Oncogene, 9:647-649, 1994) and the protein tyrosinephosphatase receptor, mu polypeptide (PTPRM, Suijkerbuijk et al.Cytogenet Cell Gene, 64(3-4):245-6, 1993) were each represented in four,three and two clones, respectively. The Gs alpha, olfactory type (GNAL,Zigman et al., Endocrinology, 133:2508-14, 1993) and 5′ H. sapienshypothetical protein (HUMKIAAN, Nomura et al. “Prediction of the codingsequences of unidentified human genes” (Genbank Accession #D42055,1993)) were represented in one clone each. In addition, the mapassignments obtained for transcripts of these five genes were consistentwith previously reported data (Table 4 and FIG. 2).

[0260] A FASTA (Pearson and Lipman, Proc Natl Acad Sci USA,85:6565-6572, 1988) sequence comparison among the remaining 37 cDNAclones to search for redundancy (defined as ∃ 389% identical sequenceover >100 bp) indicated that 20 cDNAs were unique and 17 redundant cDNAsrepresented five groups of unique sequences. Therefore, including TABLE4 Chromosome 18 specific brain derived cDNAs homologous to known genes.Clone BLASTN/TIGR Cytogenetic Percentage Identical Number sequencehomology Location 5′ 2′ Reference 1 63 kDa protein kinase related toERK3 (H863KDAP) 18q21.2-18q21.3 99.4 97.5 Li et al. 1994 3 63 kDaprotein kinase related to ERK3 (H863KDAP) 18q21.2-18q21.3 95.3 96.2 Liet al. 1994 7 Myelin basic protein (MBP) 18q23 95.8 93.5 Kambols et al.1986 10 63 kDa protein kinase related to ERK3 (H863KDAP) 18q21.2-18q21.399.6 99.4 Li at al. 1994 11 Protein tyrosine phosphatase, receptor-type,mu polypeptide (PTPRM) 18q11.2 none 89.2 Suijkerbuijk et al. 1993 12Myelin basic protein (MBP) 18q23 95.7 92.1 Kambolz et al. 1986 18 Myelinbasic protein (MBP) 18q23 94.5 99.1 Kambolz et al. 1986 31 Guaninenucleotide-binding protein, alpha-subunit, olfactory type (GNAL)18q11.22-p11.21 98.9 98.6 Zigman et al. 1993 40 5′ H sapienshypothetical protein (HUMKIAAM) 18q21.3-18qter 99.4 none Nomura et al.1995 43 Protein tyrosine phosphatase, receptor-type, mu polypeptide(PTPRM) 18p11.2 94.0 90.0 Suijkerbuijk et al. 1993 46 Myelin basicprotein (MBP) 18q23 95.3 none Kambolz et al. 1986

[0261] the transcripts for the known genes, we have identified a totalof 30 unique transcripts, of which 25 did not exhibit homology topreviously known genes. The insert sizes of the cDNA clones that weredetermined to be chromosome 18-specific ranged from 1 to 2 kb (Table 3).To explore the presence of an open reading frame (ORF) in each clone andto further examine any homology to known genes, we determined theremaining sequence of the unique clones (sequences were deposited in theGenbank, with the following accession numbers: U55777 and U55962 toU55991). We found potential polyadenylation signals in some of theclones. So far, no ORFs have been detected suggesting that a majorportion of the cDNA clones corresponded to 3′ UTRs. More importantly,comparison of the longer sequences of the cDNAs with sequences in thedatabases failed to reveal significant homology with any known genes,supporting the idea that these transcripts were derived from novelgenes.

Example 11 Radiation Hybrid Mapping

[0262] To achieve a higher resolution map for each of the transcripts byPCR, we used the Stanford G3 radiation hybrid series and primersspecific for each cDNA. Of the 25 unique transcripts, 19 weresuccessfully linked to chromosome 18 STSs (see Table 5, below and FIGS.2 and 3).

[0263] The positions of the cDNAs in the radiation hybrid framework mapwere consistent with their subchromosomal assignments (FIGS. 1, 2, and3). With this method, fine mapping was established for the uniquetranscripts as evidenced by the physical distance between them and thechromosome 18 STSs which ranged from approximately 4 to 46 cR, estimatedto be between approximately 100 kb and 1100 kb (FIGS. 1, 2, and 3).

[0264] Radiation hybrid mapping was also used to position the knowngenes identified in this study against the 25 non-redundant transcripts.We found that HS63 KDAP formed a high resolution linkage group withclones 2, 4, 19 and 33 (Table 5 and FIG. 2). The fine map locations ofthe anonymous markers D18S37, D18S53 and D18S40 were similarly examined,since they showed excess allele sharing in manic-depressive affectedsib-pairs in two studies (Berrettini et al., Proc Natl. Acad Sci USA.91:5918-5921. 1994; Stine et al., Am J Hum Gene, 57:1384-1394, 1995).These three markers. GNAL and cDNA clones 22, 24 and 37 assembled into aseparate radiation hybrid linkage group (Table 5 and FIG. 2). Furtherinvestigation into the linkage overlaps between the STSs, genes andTABLE 5 Radiation hybrid mapping of unique cDNA clones* Test ReferenceDistance to Reference Marker Marker LOD Marker (cR8000) 14 D18S476 10.225.78 D18S481 13.3 15.04 D18S54 8.9 32.31 D18S63 10.9 22.50 D18S459 10.225.78 D18S1132 7.0 42.16 34 D18S476 8.3 32.61 D18S481 12.4 16.08 D18S5412.7 15.96 D18563 24.6 9.61 D185459 14.6 9.61 14 7.6 38.31 D18S1132 6.841.79 24 D18S464 11.0 16.07 D18853 6.1 45.87 GNAL D18S4B2 7.8 36.15D8S71 9.0 31.62 D18S53 D18S464 7.5 35.77 D18S482 7.0 39.77 D18S71 6.942.39 37 D18S73 7.1 40.23 D18S71 6.0 38.03 22 D18S73 7.7 37.31 D18S4013.3 26.92 D18S40 D18S73 11.2 21.40 D18S71 7.3 40.07 D18837 D18S73 13.516.28 D18S71 7.6 39.76 39 D18S1101 7.8 37.31 13 D18S1160 7.8 28.72D18S475 7.7 19.22 25 D18S454 10.3 10.73  8 D18S460 7.8 28.65 D18S72 7.828.72 15 D18S460 7.2 30.83 D18S72 8.5 24.10 8 12.5 8.60  4 D18S470 6.142.49 19 6.6 35.80  2 19 14.3 4.08 D18S470 12.6 11.47 4 6.0 40.67D18S474 8.3 24.21 D18S69 8.2 27.02 HS63KDAP 2 10.5 17.02 D18S474 9.818.18 18 9.1 22.46 D18S470 7.7 33.44 D18S69 6.5 38.01 19 D18S470 10.319.96 D18S474 8.4 24.51 D18S69 7.1 33.41 33 D18S69 7.1 33.41 41 D18S4366.5 33.52 D18S53 7.2 30.83 23 15.1 3.85 23 D18S58 6.6 35.71 43 23 14.47.28 41 12.7 11.44 D18S58 6.5 35.88 MRP D18S554 7.8 25.97  5 D18S70 7.021.37  6 D18S70 6.3 28.01  9 NL 16 NL 29 NL 30 NL 36 NL 47 NL HUMKIAANNL PTPRM NL

[0265] unique transcripts showed that at least six radiation hybridlinkage groups were evident (FIG. 2). Based on these physicalrelationships, a map order within each linkage group could be deduced.

[0266] In sum, using direct cDNA selection and physical mapping by PCR,we have identified and positionally catalogued 48 chromosome 18-specificcDNAs that are expressed in infant brain. Sequence database comparisonrevealed a level of redundancy in the 48 clones, yielding a total of 30unique transcripts. Five genes previously assigned to chromosome 18 wererepresented in these transcripts. Additional sequence analysis of theremaining 25 non-redundant cDNA clones and database comparisons failedto elicit any significant homology to known genes indicating that thesebrain-expressed transcripts represent novel genes.

[0267] So far, we have no evidence for possible redundancies among theunique transcripts due to alternative splicing or the presence ofpseudogenes, but these probably are very minor components of the cDNAlibrary. Polymeropoulos et al., Chromosomal distribution of 320 genesfrom a brain cDNA library, Nature Gene (1993) suggested the possibilitythat chromosome 18 may be gene-poor. A recent effort to sequence and mapcDNAs yielded only four on chromosome 18 out of the several hundredcDNAs localized to other chromosomes (Berry et al., Gene-basedsequence-tagged-sites (STSs) as the basis for a human gene map, NatureGene 10:415423 (1995). The 25 unique cDNA clones isolated in this study,therefore, represent a significant increase in the number of new geneson chromosome 18.

Example 12 Transmission Equilibrium Test (TDT) on Clone 22 in TwoPedigree Series

[0268] In the pedigree series described in Berrettini et al.,Psychiatric. Gene. 2:125-160 (1991), incorporated herein by reference)linkage disequilibrium with manic-depressive illness is observed forgenes within the region of the radiation hybrid map (FIG. 3) betweenmarkers D18S843 and D18S869. The best results are given by clone 22,where allele 2 shows preferential transmission in this and a secondindependent pedigree series (see Table 6, below).

[0269] The second pedigree series is the manic-depressive pedigreeseries recently made publicly available by the Nationals Institutes ofMental Health as part of its Genetics TABLE 6TRANSMISSION/DISEQUILIBRIUM TEST (TDT) ON CLONE 22 IN TWO PEDIGREESERIES Allele Freq Transmitted Not Transmitted P-value Bethesda BipolarPedigree Series 1 0.344 37 59_(*) ^(S,) 0.025 2 0.656 59 37_(S−) ^(*)NIMH Genetics Initiative Collaborative Series 1 0.376 83 116 0.019 20.622 118 83 0.014 3 0.002 0 2 N/A

[0270] Initiative. The statistical test is thetransmission/disequilibrium test (TDT) of Spielman R. et al., Am. J.Hum. Gene. 52:506-516(1993). In the analysis of these two pedigrees,manic-depressive individuals are genotyped to determine the whether aparticular allele of the clone 22 polymorphic marker has beentransmitted or not transmitted to them. Given the relative frequenciesof the alleles, the probability (P-value)of the co-occurrence ofmanic-depressive illness and a particular clone 22 allele is determined.The results indicate preferential transmission of allele 2 tomanic-depressive affecteds.

Example 13 Discovery, Characterization and Isolation of IMP.18p

[0271] This example sets forth the discovery, characterization andisolation of the novel inositol monophosphatase gene and protein of theinvention, designated IMP.18p.

[0272] Linkage of manic depression/bipolar disorder to the broadpericentromeric region of chromosome 18 (Berrettini (1994) supra; Stine(1995) Am. J. Hum Genet. 57:1384-1394) motivated a search for novelgenes and (gene products which are associated with this disease. Theinitial bipolar disorder-chromosome 18 linkage region spannedapproximately 40 centimorgan (cM). Genetic analysis in the 22-pedigreeseries, reported by Berrettini (1994) supra, indicates that the highestallele sharing is in markers mapping to 18p11.2. An association (nominalP<0.05) was found at either D18S53 or D18S37 (designated S53 and S73(S37 and S73 amplify the same locus), respectively, in Table 3) on18p11.2.

[0273] As described in Example 11, above, markers within 18p11.2 showingincreased sharing were mapped using a radiation hybrid (RH) panel to anapproximately 6 megabase (Mb) region. These lines of evidence indicatedthis region on 18p11.2 is a site for the identification of transcriptsand genes associated with bipolar disorder. Electronic databanks weresearched for ESTs, sequence tag sites (STSs) and genes which had beenmapped as being encoded in the general area of 18p11.2.

[0274] This search identified a human STS of 145 base pairs, identifiedas A006N05 (GenBank), localized between D18S464 and D18S71, markers thatmap to 18p11.2. This STS had been isolated and mapped by The Instituteof Genome Research (TIGR) and was included in an approximately 1 kb TIGREST, designated contig THC98649, described by Boguski (1997) NatureGenet. 10:369-371, http://www.ncbi.nlm.nih.gov/UniGene/index.html.

[0275] The STS sequence of A006N05 was searched using the transcriptdatabase of Schuler (1996) Science 274:540-546,http://www.ncbi.nlm.nih.gov/SCIENCE96/, and, updates in Unigene athttp://www.tigr.org/tigr_home/index.html, of the National Center forBiotechnology Information. Using the method (described in Altschul(1990) J. Mol. Biol. 215:403410), it was discovered that the humanTHC98649 EST contig contained an upstream sequence exhibiting about 60%nucleotide homology (sequence identity) with the inositolmonophosphatase (IMP) of Xenopus laevis. Manipulation of these databanksfurther discovered that the THC98649 EST contig is included in theapproximately 1.2 kb insert of the IMAGE human cDNA clone ID #39740(I.M.A.G.E. Consortium, Human Genome Center, DOE, Lawrence LivermoreNational Laboratory, Livermore, Calif.).

[0276] To identify which human cells express this or a message closelyrelated to clone ID #39740, Northern blots of various human tissues wasperformed. Northern blots of multiple human tissues were purchased fromClontech (Palo Alto, Calif.). The Northern's probe was prepared byamplifying the insert of the cDNA clone #39740 with M13 forward andreverse primers, then ³²P labeling the amplified product using astandard random primer method. Blots were hybridized using Rapid-hybbuffer (Amersham, Cleveland Ohio) at 68° C., with 2×10⁶ cpm/ml probe.The final wash was done at 68° C. with 0.1×SSC containing 0.1% SDS. Theblots were exposed onto X-ray film overnight at −70° C. withintensifying screens.

[0277] The amplified probe from cDNA clone ID #39740 was found to detecta major band of approximately 1.5 kb in multiple tissues throughNorthern hybridization, as shown in FIG. 4. FIGS. 4A, 4B, and 4C showthat this cDNA probe hybridizes to a 1.5 kb message to varying degreesin: fetal brain, lung, liver and kidney; adult heart, brain, lung,liver, skeletal muscle, kidney, and pancreas; and, adult brain amygdala,caudate nucleus, corpus callosum, hippocampus, hypothalamus, substantianigra, subthalamic nucleus, and thalamus. Control hybridizations weredone with GAPDH (constitutively expressed in all of these tissues). Ascan be seen by the relative intensities of the hybridization bands ascompared to control. IMP.18p was abundantly expressed in both adult andfetal skeletal muscle, pancreas, heart, placenta, liver and lung, but toa more limited extent in whole brain. Contrary to the minimal level ofIMP.18p transcript in whole brain, a substantial expression was found inbrain subcortical regions with caudate showing a higher expression levelthan the other anatomical substrates (FIG. 4C).

[0278] cDNA clone #39740 was sequenced by conventional techniques.Analysis of this sequence showed that clone #39740 was missing its5′-end coding sequence. Additional upstream coding sequence was acquiredusing rapid amplification of cDNA 5′ ends (5′ RACE) PCR, as describedabove. Marathon-Ready cDNA derived from human skeletal muscle (Clontech,Palo Alto, Calif.) and the clone-specific primer designated p1:5′-ACGTCGGGCTGTGGGTGAGCACACACTTG (SEQ ID NO:24) (corresponding tonucleotides number 405 to 433 of clone #39740). PCR was performed usingan initial one minute denaturation at 94° C., followed by 5amplification cycles at 94° C. for 15 sec, 72° C. for 2 min; and, 30cycles of 94° C. for 15 sec, 65° C. for 30 sec, 72° C. for 2 min, andfinal extension period at 72° C. for 5 min, using Taq DNA polymerase(Perkin Elmer) and MasterAmp 2× PCR PreMix I (Epicentre Technologies,Madison, Wis.). Sequencing was conducted using a dye terminator cyclesequencing kit with Taq FS (Perkin Elmer Applied Biosystems, FosterCity, Calif.) and the ABI 373 DNA sequencer (Applied Biosystems, supra).Each nucleotide sequence was verified using at least two independentsequence reactions including both strands. Sequence similarity search,alignment and motif detection were done using the Genetics ComputerGroup, Inc. (GCG, Madison, Wis.) computer package.

[0279] The RACE method extended the upstream region of clone #39740 by278 base pairs and included the potential initiation methionine. FIG. 5Bshows the complete 1447 base pair full-length cDNA nucleotide sequence(SEQ ID NO:16) and the corresponding predicted amino acid sequence (SEQID NO:17). An in-frame stop codon in the 5′ untranslated region (UTR)and a poly(A) signal in the 3′ UTR are underlined. FIG. 5A shows aschematic representation of this newly discovered message aligned withclone #39740, the open triangle depicts the coding region. Also shown asarrows are location of the sequence used to design primers p2 and p3used in radiation hybridization mapping, discussed below.

[0280] The location of this IMP gene on chromosome 8 was establishedusing ESTs from the Unigene databank (Boguski (1995) supra).

[0281] The 1447 bp full-length cDNA has a predicted open reading frameencoding a protein with 288 amino acids and a G-C rich 5′-untranslatedregion (UTR) (FIG. 5B). A protein homology search (as described inAltschul (1990) supra) showed a 53.5% identity to a human brainmyo-inositol monophosphatase (IMP) gene, as described by McAllister(1992) Biochem. J. 284:749-754. The McAllister, (1992) supra, IMP cDNAhas a considerable sequence difference and is encoded in a distinctchromosomal localization as compared to the IMP of the invention. Thus,this newly discovered IMP represents a novel gene and protein, and isdesignated IMP.18p. As shown in FIG. 6, IMPs protein sequences fromXenopus laevis (SEQ ID NO:25), rat (SEQ ID NO:26) and bovine (SEQ IDNO:27) have a 54.8%. 53.5% and 53.8% sequence identity, respectively,with the IMP.18p protein. Xen, Bov and Hum represent Xenopus laevis,bovine and human sequences. Dots indicate identical amino acids. Infurther contrast to other IMPs, as shown in FIG. 6, the IMP.18p of theinvention has an additional 11 amino terminal residues not seen inpreviously characterized inositol monophosphatases.

[0282] Two protein motifs characteristic of the myo-inositolmonophosphatase protein family, which includes animal inositolphosphatases, fungal and bacterial regulatory proteins of unknownenzymatic activity as found by Neuwald (1991) FEBS LETT 294:16-18, werealso found in IMP.18p, as indicated in FIG. 6. Motif A has the consensussequence (W)x(I)DP(I)D(G)Tx{2}(F)x(H) and motif B has the consensussequence: Wdx{2}(A){2}x(V)(I){2}x{3}(G,A){2}.

[0283] In IMP.18p, motif A and motif B correspond to amino acids number98 to 111 and 230 to 244, respectively, see FIG. 6: as numbered in FIG.5B. In IMP.18p, the amino acid residues Asp101, Ile103, Asp104, Thr106and Asp231 (as identified by the base pair numbering in FIG. 6) fallinside the motif regions characterized by Neuwald. These motifs havebeen suggested to exert an important role in metal binding (see Pollack(1994) “Mechanism of inositol monophosphatase, the putative target oflithium therapy” Proc. Natl. Acad. Sci. USA 91:5766-5770) and in thecatalytic activity of the human IMP on chromosome 8 (see Pollack (1993)“Probing the role of metal ions in the mechanism of inositolmonophosphatase by site-directed mutagenesis,” Eur. J. Biochem.217:281-287). Sharing these structural motifs, IMP.18p is also expectedto have inositol monophosphatase and lithium binding capabilities.

[0284] Northern hybridization was conducted under high stringencyconditions to minimize cross hybridization with homologous mRNAs (i.e.,wash conditions that minimized cross hybridization were used: 0.1×SSC,0.1% SDS, 65° C.). The human chromosome 8 IMP of McAllister, (1992)supra, expresses a transcript that is 2.2 kb (Pollack (1993) supra),which distinguishes it from the novel IMP of the invention, IMP.18p,whose primary mRNA transcript, as determined by Northern blot, is 1.5 kbin length (see FIG. 4).

[0285] To achieve a fine physical localization on chromosome 18, IMP.18pwas further mapped utilizing radiation hybrid (RH) mapping (using theStanford Human Genome Center's (SHGC) G3 panel, as described by Cox(1990) “Radiation hybrid mapping: a somatic cell genetic method forconstructing high resolution maps of mammalian chromosomes.” Science250:245-250), as described in Example 11, above. Multipoint RH mappingdetermined locus order and interlocus distance between IMP.18p and othermarkers on 18p11.2, using the MultiMap/RADMAP computer programs (asdescribed in Matise (1995) “Automated construction of radiation hybridmaps using MultiMap,” Am. J. Hum. Genet. (Suppl.) 57:A15), under anequal retention model. Strategies for multipoint RH mapping andselection of markers has been described, for example, in Lunetta (1996)Am. J. Hum. Genet. 59:717-725; Francke (1994) “A radiation hybrid map ofhuman chromosome 18,” Cytogenet. Cell. Genet. 66:196-213, also,http://www.ebi.ac.uk/RHdb/vers_soft.html. Initially, a linkage groupwith the criteria of a lod 5 and a breakage probability of lod 0.3 wasidentified. Next, markers from the linkage group were mapped with aplacement threshold of lod 3. This analysis ordered 11 loci in a regionof 175.4 cR (approximately 4.7 megabase. assuming the mean ratio of 26.8kb/cR in chromosome 18 (Cox (1990) supra) and placed IMP.18p betweenguanine nucleotide-binding protein-olfactory, type-a subunit, or G(olf),(“GNAL,” Berrettini (1990) supra) and D18S71. This mapping positions thegene encoding the IMP.18p myo-inositol monophosphatase of the inventionwithin the bipolar susceptibility region at 18p11.2, as shown in FIG. 7.

Example 14 Characterization of the Promoter Region of IMP.18p

[0286] This example sets forth the discovery and characterization of thepromoter region of the novel inositol monophosphatase gene, IMP.18p, ofthe invention.

[0287] To facilitate screening of the entire IMP.18p genomic sequenceand to provide for a monophosphatase-specific transcriptional regulatoryelement for use in the construction of, for example, tissue-specificexpression vectors, transgenic animal expression cassettes, and targetsfor expression-regulating nucleotide sequences, the promoter of IMP.18pwas identified and characterized.

[0288] Cosmid clones from a chromosome 18-specific cosmid libraryLL18NC02, from Lawrence Berkeley Laboratory, were isolated by spottingthe library onto nylon membranes to generate high density filters. Thesefilters were hybridized with a IMP.18p cDNA probe (SEQ ID NO:16). Threeclones, designated 119C4, 97A4 and 69E10, which hybridized to the cDNAprobe were isolated. Sequencing was performed using the dye terminatorcycle sequence kit with TaqFS from Perkin Elmer-Applied Biosystems, Inc.(ABI, Foster City, Calif.) and an ABI 373 DNA sequencer.

[0289] The transcriptional initiation site was determined by primerextension using a “Primer Extension System” from Promega (Madison,Wis.). An IMP18.p-specific antisense oligonucleotide primer (underlinedcoding sequence in FIG. 8, designated “p”) was 5′ end-labeled with gamma32-P ATP and T4 polynucleotide kinase. 100 fmol of the labeled primerwas annealed to 1.6 ug of poly(A)+ RNA derived from skeletal muscle(Clontech) at 58° C. for 20 minutes, and then kept at room temperaturefor 10 minutes. Annealed primers were extended with AMV reversetranscriptase at 42° C. for 30 minutes. The extended products wereanalyzed on a 6% sequencing gel, electrophoresed beside a sequenceladder that was generated by sequencing the appropriate region of cosmid97A4 with the same primer used in primer extension analysis 5′-GGG CGACCG ACG GGA AG-3′ (SEQ ID NO:28).

[0290] The major extension product was 183 base pairs (SEQ ID NO:29)corresponding to 160 base pairs upstream of the initiation ATG. as shownin FIG. 8 (the nucleotide sequence of the 5′ flanking region islowercase, and the upstream portion of exon 1 is uppercase). The majorcap site is indicated as nucleotide +1 and is denoted by an arrowpointed in the direction of transcription. A minor transcriptional startsite, as shown by primer extension analysis, is a “T” residue atposition (minus) -6. The translational initiation codon is boxed.

[0291] The sequence around the transcription initiation site did notindicate the presence of TATA and CAAT boxes. However, there weremultiple, potential recognition sites for Spl, in addition to consensussites for other transcription factors, as indicated in FIG. 8. TATA-lesspromoters have been described in “housekeeping genes,” oncogenes, growthfactors and their receptors, and transcription factors (as reviewed inAzizkhan (1993) Crit. Rev. Eukaryotic Gene Expression 3:229-254). Thepromoter region of IMP.18p gene has several features shared by otherTATA-less genes, including a GC-rich sequence with multiple CpG islands;several Spl consensus motifs; and, heterogeneity in transcriptioninitiation (FIG. 8).

[0292] All publications and patents mentioned in this specification areherein expressly incorporated by reference into the specification forall purposes to the same extent as if each individual publication orpatent was specifically and individually indicated to be incorporatedherein by reference. (Upstream) SEQ ID NO: 1 (Downstream) SEQ ID NO: 2Clone 22 primer sequence: upstream primer: 5′-CAA GTT TAT GTT ACT GCCAGG G-3′ downstream primer: 5′-GCA GCT TOC TAA TGC ATC CAG-3′ (unsplicedprotein) SEQ ID NO: 3 (alternatively spliced protein) SEQ ID NO: 4(spliced portion) SEQ ID NO: 5 1 MPEAGFQATN AFTECKFTCT SGKCLYLGSLVCNQQNDCGD NSDEENCLLV 51 TEHPPPGIFN SELEFAQIII IVVVVTVMVV VIVCLLNHYKVSTRSFINRP 101 NQSRRREDGL PQEGCLWPSD SAAPRLGASE IMHAPRSRDR FTAPSFIQRD151 RFSRFQPTYP YVQHEIDLPP TLSLSDGEEP PPYQGPCTLQ LRDPEQQMEL 201NRESVRAPPN RTIFDSDLID IAMYSGGPCP PSSNSGISAS TCSSNGRMEG 251 PPPTYSEVMGHHPGASFLHH QRSNAHRGSR LQFQQNNAES TIVPIKGKDR 301 KPGNLV (comprising SEQID NO: 7, coding far protein of SEQ ID NO: 6 SEQ ID NO:3) and (codingfor protein cf SEQ ID NO: 4) ) SEQ ID NO: 8 Clone 22 common regionnucleotide sequence Length: 8065 CCCAGCAGAG CGATGGACTT GGACAGGCTAAGATGGAAGT GACCTGAGCC 51 TCGCCCGCCG GCTTCCTCGA CGGGACAGCG CAAGAGTTGGAGCACAGGCT 101 TGTCCGGGGA GCAGTATGCC GGAAGCTGGT TTTCAGGCCA CAAATGCTTT151 CACAGAGTGC AAATTCACCT GCACCAGTGG TAAATGCTTG TATCTTGGTT 201CGCTGGTCTG TAACCAACAG AACGACTGTG GGGACAACAG TGACGAAGAG 251 AACTGTCTCCTGGTGACCGA GCACCCGCCT CCGGGCATCT TCAACTCGGA 301 GCTGGAGTTC GCCCAAATCATCATCATGGT CGTGGTGGTC ACGGTGATGG 351 TGGTGGTCAT CGTCTGCCTG CTGAACCACTACAAAGTCTC CACGCGGTCC 401 TTCATCAACC GCCCGAACCA GAGCCGGAGG CGGGAGGACGGGCTGCCGCA 451 GGAAGGGTGC CTGTGGCCTT CAGACAGCGC CGCACCGCGG CTGGGCGCCT501 CGGACATCAT GCATGCCCCG CGGTGGAGGG ACAGGTTCAC AGCGCCGTCC 551TTCATCCAGA GGGATCGCTT CAGCCGCTTC CAGCCCACCT ACCCCTATGT 601 GCAGCACGAGATTGATCTTC CTCCCACCAT CTCCCTGTCC GACGGTGAAG 651 AGCCACCTCC TTACCAGGGGCCCTGCACCC TGCAGCTCCG GGACCCTGAA 701 CAGCAGATGG AACTCAACCG AGAGTCCGTGAGGGCCCCAC CCAACCGAAC 751 CATATTTGAC AGTGATTTAA TAGACATTGC TATGTATAGCGGGGGTCCAT 801 GCCCACCCAG CAGCAACTCG GGCATCAGTG CAAGCACCTG CAGCAGTAAC851 GGGAGGATGG AGGGGCCACC CCCCACATAC AGCGAGGTGA TGGGCCACCA 901CCCAGGCGCC TCTTTCCTCC ATCACCAGCG CAGCAACGCA CACAGGGGCA 951 GCAGACTGCAGTTTCAGCAG AACAATGCAG AGAGCACAAT AGTACCCATC 1001 AAAGGCAAAG ATAGGAAGCCTGGGAACCTG GTCTGATTCC TTCCAACGTG 1051 CACTTCAGCT GGAGAAAGAA ACCAAGAAGGGAAGCGGCCG CTGGGCCCCT 1101 CCTGGGCACA GTGTTGTTCA GTTTCACATG GTACAAATAAGTAAAACCAA 1151 ATGAGCAAAC ACGGTCTTTG TTTCTGATTC CTTTTAGGGG AATTGCATGC1201 AAACTAGACT GAAATGATAC AAACTTCCAT CTGGTCTGAC CGCAAACAGT 1251GTTTATTTGG GGACAGGGGT TGGGATGGGG GTGTGGGCAG GGGAAAACAG 1301 AGAACGGGATGCTTTGAAGA TACCATGAAA TAAAACCCAC AGAGGTATTT 1351 GATGTATTTA ATTGTGAAAGGAGACTTTGC AGATAAATGA GGCCAGAATG 1401 GCATGTTTTA TAATTAACTG AATAAAGAAGGAAGCATTAT TATATATTAT 1451 TGTGGGGAAG AACCAGCCAG TTCGCTTTTT CTCCTAAGGTGTGGACTTTT 1501 ATTTTGTTTT AAAAATATGA ATCAAAATTC CTGTGTTGTG TGCCAAGGTA1551 TAAAGTGGAG AAGTTAGATG AGTGCAAGGA GCTCCTTTGT GTTGTGATGA 1601TGTGTTTTAA AAGTTGCACT ATCTTAATGT TGAAAATATT TACAAGGGAA 1651 CTGTTTTACGTGAAGTTCTG TATGTTGTCT TTTCACCTGT GGATTGTAAT 1701 CAGGCCCAAG GAATATCCTGGAGTGGTCCC CAGAAGCATC CAAGAAAAGA 1751 TATTTGGGGA CGTAGCCTAA CATTTTACCAACTTACGTAA ATCAAAAAAG 1801 TCATTATTGT TGCAGGAGTT TGCATCAAAT AGCAGTGCATCGCTGAAGCT 1851 TTTGGAGACT TTTGGATGGA AGATAAGATA GGGAAGATTA AGTTCCAGCA1901 TTTCTGACTT GTTATTTTGA GTTACTCTGC TACTCTTAGG CTGCATAGTT 1951TATGAGAAAA TGAACACATG CATTTATGGA TCCAGTATCA TGCAGTGCTG 2001 CCCTCATCCTCCAGCAGTGC AATTTCTTCA GTAATTTAGA TTTTTTTCAC 2051 TATAGCATGA AATATATTCAAATACATACC TTATTTTATG CAATAAATTG 2101 TTTAAAATGC AAGGTGGTTA TTCTGCATACTGTTGAAATA TGTGACTCCT 2151 CAGTATATTC CCATTGCCTC TCCCCCTTTC CTCGACAGCTTAGTTCAGTT 2201 CTGCAGGGCT GCTCAGTTCA CAGGAGGCTC CCAGCAGCCA CCCCACATCC2251 AGCCTACACA GAACTTTCGT GTGGGAGTGG TGTGGGTGGT GGTTTTCTTA 2301TGCTTTGGAA GCCCCTAGAA ATAATGACGG AAGAATGCCA TGTTGCTGAT 2351 CGTGGTAATAAGCCATTGTG GGTTATTGTA TGTCACTAGT ATTAGCATAG 2401 CATTCTTAAA GGAATGCAGTGTTCAAAACC TACCCAAATT CCCCGCAGGA 2451 TTTTACCAAA CCCTTCCCCA GGCCAGTTTTGTACTGAAGG CAAGAACTGG 2501 ACAGTCAGAG AACAGTGGAG GGGGCAAGTG ACTGAAGAGCACCGGGTAAA 2551 AAGCACAACA TGCAGTTAAA ATGCAAACTA GAAAACTAAT TTTAAATATT2601 GTTAGTTTTA ATATTTCCTG ATATTTACAA ATATTCATTC TTATATACAA 2651TGAAAAAAAT AACTTTCTTC TGCAGATGTA AGCACTGGCT TTTATAAGAG 2710 CAGCAGCCAACACGTTTAGC AGACACTGCG CGTGGAGAAG GGCTTATCTG 2751 CAGTACACTC TGCCATGTGGAGGGTGGGCC TCTGTGGCCT CTTCACATAA 2801 CAAGATGAGC TGGAATGATG ATTCCATGACTCCCACCTAT GCAGCCTTAA 2851 AGCCAAATCC GCGTGTGTGT GTTTGTGTCT GTCTGTGGGTCTCGAAGGTG 2901 ATCCGTCGGT GCGGTGGCTC TGTGCTGTAA CTGGAGAGAC TGTTCCAAAC2951 CCCAAGAGTT GTCTGATCCT AGTCTGTTCC CTTCTGCTTC TTACCTCTGT 3001AGATAGGTCA CTGGTTTTTG TTTGTTTGTT TTGAGGATTG GAATTTCCAT 3051 TACATTCATCCTTTGCACAC AGTAACATCC ACAGAACTAG TCCAACTCTT 3101 AAAAGGAGAG AGGAAAAACACAGGCACCAG TTGTCAGCTC ATCGTTACAA 3151 CCTGTGTGGA AGTATATACA GTTGAGAGTCACAGTGGAGG TTCTGAGACT 3201 GGATTCAGTC TTGTTCCAGT GACAGTTGGA AGGCCTCTGCTGGAGAGACA 3251 CCAGCTCTCA GGGCAGAGAT TGGCTTGGGG CCAGAAGGAC CCTCCCCAAC3301 CCTGGAGACA CCCTGAAGGT TCACTGGCTC TCCAGATTAG CCTCTCTTCC 3351TCTGTCAGGC AAAGATGAGG AGCCCGTGTT CCCATCGGGC CCTGCTGGCA 3401 GGGACTTGCAGTGGATTCTT GGTCAGGTGT GCCCACAGAT GCGGAGGCGA 3451 GGTGAGTGAT TCCATCATTTCAGTTCTCAC CTGCAGTTTT GGTGAAGCAG 3501 GAGATGCACC CCACAGCTCT AGCTCTCAAATGGCTTCACA GTCCTTACTT 3551 CTCTACCTGC CTCAAGAAGG GGCTCAGAGC AGAGACTTGTGAATTCCTTA 3601 GTAACTGTGA GTATATGAAT GTGTTGCACA TGTCCACAGT ATTGGCCAGA3651 TAATTACATA ATTCAGATAC CTTTAATCAT CTTTCAAGAA AGAGGCTCCT 3701CCCATTCAAC CACCCTAGAG AACTGCCTTT GTTAAATAGT TATTTAAAGA 3751 CTCATACATATCAAACCATG ACTTTGAAAG GTCTTCGAGG CTGGGGCTCT 3801 GTAATGAATT AGTTTAAAAGCCAAGGTCAT AACATGAATT GATGGTCAAT 3851 TTCCCTTCAG CAGAAGGAAA AGGTGATTTAGATCAGTAGC TCTTTTGAAG 3901 GTTGTGGCTG ACCTGTTCAT ACCGTGTCGC CTCATGGCTAGTGTGGCGTT 3951 GAAAGAGTAG CGACTGGGAA GATACAACTT ACACAGTGGG GCCTATTGTT4001 CTTTCAAGAA CCCTTTTTTT AGCTTATAGA ACCCATGGGT CCAGTTTAGT 4051AACGAGTGAT TTAGGCAATC AATGATAGGT TTATAATCTT AGATTATTCC 4101 AGCAAAGTGTGGATTGCATT GTTAGGAAGA ACATTTGGTG GGAATGAACA 4151 CTCCTGGGCA TACCGCTGACTTTTGTCCCT TGTTCCCGGT GTAGGAGACC 4201 CAAGGCATCT TGAATCCCAT CTATAAGAACACAATCTTCC AGCATACGTT 4251 TGCTTTTTCA GAAACTCTAG CATTCTCTTT AAATACTGACGCAATCCTTA 4301 ATGGAAAAGA GATTTCATGA AGCAAATTAT GTATTTCAAT AGTTCTTCTA4351 TTTTTAGTGT CCAAAATTTA CTAATACAGA AGCTTGACAA GCATGTCCTC 4401ACCCTCCCCA CCACATAAAC ACATGGACAC ACACCCAAGC CACAAGAAAT 4451 CCCAAGAGAGCAGAAGCGAA TTTTTAAAAG ATTTATCGTG AGGACTGCAT 4501 TTCCATTCAC TAATTTTGGCTCAAACTTAT GAGGCAGGAA ATAGGGGCCA 4551 ACAGTAAATG GGGGAGGCCT CCTGACACCAGCAGAGGAAT TTTGTACCCA 4601 GGCGAGCACT TCTTGAACTT CTGCGTATCT CCGTTTGATCTCTTTCACCT 4651 TTATTTCATC TTCATAAGAA TGAGAAAGGC TCAAAAGGAA GCACTTTTAG4701 AAATCTTCTC TGACCTAGAA GAATCCATCC AAATCCCTGC CTTCCTCTCT 4751GAACCAACAG TTCCCTTCTC TGACAGGGGG CCATCCTCTA TATTCCATCC 4801 AGCGGCTCTTCCTTTTAGGA AGGCTCTGGT GCAGAGCACT TCAAATATGT 4851 CCTCAGGCCA GATACTGATTGCTAGTAGAG AGACACCCGG CACCCAGTCC 4901 GAAGCCCTCC CTCAAAGGAC CGGCTTATGGCGTTGGTCAC TGGCAGGCTC 4951 AGAGACATTC TACTGTGGGC GCAGGGAGCC CGGCCCCCCATGCAGCCATG 5001 ACTGGATGCG CCCCCATCTC GGGGGCTTGC TGCACTGCTT GTTTATTGAA5051 TTTTGCTACT TAGAATGGCA ACATTAACTT TGTGTACCAT TCATTTTTTA 5101AAAATTTTCC AAAGCTCGGC AGTGTATGAA AGAAAAAACT GGGAAAGATA 5151 CTTGGTTTCTGTTAACTTTT GTGTTGCTTG CTTAAGTGAT TAAAGCCAGT 5201 GCTTGGAGCC AAGCCTTCATGCCACGAACA TGCTCCACAG CCTGCCCTTT 5251 GCTCTCCTGC TCACACTGAC CAAGAATGCCGCGTGCTTGG CCTACTGAGG 5301 TGAAAGGACA ATTGAATGAC AGGTGGGCAA AGGGAGAACTTCCCCTTCTT 5351 GGTGCGAGGA AAGTCACAAA TTTAAAAATG TTGCTTCCAG CCCAGATCCT5401 AAATGCTAGT TCTCAGCAGC TGCGTGGCTT ACCGTTCGCC ATTTCCACCA 5451CCGCCAGCTG CCAGCACCGC TACAGATCAC AGAGATGTGA ACAGACAATC 5501 GAAAGCACTCTTAGCCTTGC AGTGGTCTAC ATTTTTTAGG AACCAATATT 5551 TCAGCATTCT TTATTACCCGGCACGCTGTG TCCTTTGCAG AGTTCAAGTT 5601 TATGTTACTC CCAGGGTCAG ACAGTCATTTGTCGCTGCTG CTGCTGCTGC 5651 TGCTGCTTCT CGAACTGGAT GCATTAGGAA GCTGCTGTCTGAGTGTAGGA 5701 ATGTCTTGCT AAGAAAGCAA TGTCTTCCTT CATCCTTTTC TTTCTTCCCT5751 CTGCGTGTCC TTGTTTTTGT GTAATGCGGG AGAGGGTTAG AGCTATAGAG 5801ATTATATATA CACTATCCGT GCACATTATA TATATGTAGA TATACCCCTA 5851 TCATGTCAGAGATCTGCATG TCAGTTTTTC AGCAACTAAG GTGCCTCATG 5901 TTCTGAGTTC AGCAGATATACGAACCAAGC CGCCCCCTCC TGCACTTGAT 5951 GCTCCCACCT TTGTTGTGCC TCACTTAAAATGGTGCTTTT TTCAGTTGTC 6001 TGTCTTTTCT TATGTTTTTA TTTGTAAGGT GCTGTATATAAGTTGAATAT 6051 ATTATGCACA TATCCTACCC AATGGGTAGA ACAAAAAGTT GTTAATACTG6101 TAATATAATG TATAGATGAT ACCAATTTTA ACAGAAATGG CATAGAATTT 6151GTGAATGCCT ATGTGCTTTG TCCTCTTTTG TAAGGAAATT TGCAAATGGA 6201 TGCATACAGATTAAAGTCTA TGTAGTTTAT TTTCCTATTA AATATCAATA 6251 TTATAACACA AGAGAAAGAAGTGTGAACAA ACAAGCAACA GTTTATGACC 6301 AGCGTATATA TAGCAATGGA AAGTTGCATCTTTGCTGTGA AAACACTTTA 6351 AAGAAAATAC TTTTTAAAAA ATCCCACAGC TTTTTGGTTGCCACTAGACG 6401 CTTCTTATTT TAATCATTTT AGTAATGCTC AGCTGGACCA GTGTTAGTTA6451 TATTTGAGTC AGAAAAATGT TGTTTTTCAA CTTGCTTTAT AATCTCCTGC 6501ATCTATCTCC TGCTGTAGCA TCAyGAAGGT GTCAGGCAAC AGTGAAAAGT 6551 GCACATTTTTGTTGTTGCAG AAACTGTGTC AGAGGAATAA GTAAATCAGC 6601 CTGCAGCAGA AGACTTTGTTCAGCTCCAGA GGCATCTGTG ACCGTCTGTG 6651 TCCAAGTCTC TCTGTGCCTT TTTCTTTTACAAACTGAAGC TGTGGAGCCA 6701 ATGAAGTAAC AGTAGAGATT GTAGGGAAAG AATACCTCAGGAAAAACAAA 6751 TACACTTACA AGAAGACCCT GTTCTTAGAA AATGTGTTTA GTTATGGGTT6801 AGCACTAGAA GAGACTTGGC TGTCAGCCAG CCAAGTGAAG GACCTCTCAT 6851CCATTCCCAT TCATGTCCCA TCATAATACG GACmCAAAAA GCAAACTCGG 6901 TTTTGCCATCAGTTAGAAAT TACGTTTTGG ATTGTATATT GTTACATCTC 6951 TCTTCCAGCT TAGTTTTTAGTGTCTGATTG TGACCTCTGC ATTTATCTTC 7001 AAATACCCTA ATTTTAAAAC AAAAGAACAAGAAAAGTTTA TAACACCATG 7051 TTCACTAAAA CCACGGTTGA ATCTTGGGTG TGGGCATCCTTTCGAGTGTT 7101 GTCCATAAGA GCAGTTCGTG GAATTTTGCC CATCTGACCC ATATTATCAG7151 CTTATTCTGC CACCAGAGTA GAGTCTAATA AATTCCAAAG TTTTTATTTG 7201CTCCATGGTG TATGTTCTGA CTTTGAAAAT GTCAGATTCT ATAATCATAC 7251 CCCTAACATCCAGGAGACAA ATGACAGATT ATCTTTAAAC TGAAATTGAC 7301 TCTACAATGC AACCCTTAATGCTGAATGGA TTAAAAAAGT CAGCCCTTTT 7351 AGTATCTGTT TGAAAGGGCC GTAAAAAGTTGACACTTTTG TTGTTGTGGA 7401 TCCTGCGTGT CTAGACCCAC GTGTTGTTTC CATCGTATACTGTAGGGTGC 7451 ACCCCTTGGG ATTCATCATT AAGAACTGAG GCTCACTGTT GTCAGAAACA7501 AAGCTCCCAC CCCCCAGGTT CAACCTTGTG GGAGAACTGT TGAGCATGAG 7551AATGTTCTAG ACTCAGAGGT ACTAAAATTT GTTACCACAT CATTGCTTCC 7601 TTTCTACAGGACGAATTGAG GCTTAAACTT TACTGTTAAT GATACTGGTT 7651 CATTTTAATG TGCTTGTTGGTATGTTGCTA TTTTTCATTT CATAGCTTTC 7701 AAAAATCATG CTAATTGTAT ACTTGTCTANTTTAAGGCTA TTTTAAAATA 7751 TGTACAATAC TATTCACAGC ATTTAGTTCG TTTAATTTTTATTATAAAGC 7801 AATCTACTAA AAAAGTACAA CTGTATTTGA ACTTTTCAAT AGTTGTTTGT7851 GAGCTATGAT AATCAAAAGT CATTAAAGTC TTTTTTAACA AACATTCGTG 7901CTTACTTTTC AACATAATTC CCAGTTATAT ACAGAAAAAG ATTTCCACCT 7951 GTCACGTATCTGCCTCTTTT ACCTGAGCAA TGGTGTAGTT CTTANACCTA 8001 AGGTCTGTAA TTGCAATACTTTTAAAGAAA GAGTTGCTCT AAGTGCTGTT 8051 TGTTAGTTAT GAAAC SEQ ID NO: 9PRIMER A: 5′ATGCC GGA AGC TGG TTT TCA GG 3′ SEQ ID NO: 10 PRIMER B:5′TCC AGC TGA ACT GCA CGT TGGCT3′ (Primers for nucleic acid encodingprotein of SEQ ID NO: 3) SEQ ID NO:12 1 TGCGAGAGCC GGGCAGGTGG GCCGCGGATGCTCCCAGAGG CCGG SEQ ID NO:13 1 ATTTCCAGTA GAGGTGGATA GAGATGGTGAGCAGCATTGA CTCTCAAAAA 51 TAGGGTCCTA TGGCTGGTAA GGAGGTTGGT GCCTTCTCGAAGGGCTAGTG 101 CTGGGAAGCT TCCTTTTAAA AACGGCCCTT TCTGCCGGTT TGGCTAGCCA151 AGAATGGCAT CCTCCTCTCT GTATCTTCCC TGGAGCTTCA GGACTGAGTA 201TTGAATGACA GAGAAGGTTC TGCAAAGTCT GCACAGGGAG ACTGCCATTG 251 CATCAAGTCATGTCTGCATT CTGTATATGC GGTTCAAGCT CTACGTTCGT 301 GACATCAAAC CTCCTGTTCGGCCATTTCCG AGAACTCCCA TCAGTTTCTG 351 TATAGTGTAA AAGTTTCAGA GGCGGAGCACAGAGAGCTGC GGCTGGGACA 401 AGGAGCACCC GCGTGCAGGT GCGACCCTGC AGGATGCTGGCAGCGGCGTG 451 GCCAGGGGCG CCCGTGTTCT GAGGGCCTGA CGGCCAGCCC C (allele 1)SEQ ID NO: 14 (allele 2) SEQ ID NO: 15 Clone 22 Allele 1CAAGTTTATGTTACTGCCAGGCTCAGACAGTCATTTGCTCCTGCTGCTCCTGCTGCTGCTGCTGCTTCTCGAACTGGATGCATTAGGAAGCTGC Clone 22 Allele 2CAAGTTTATGTTACTGCCAGGGTCAGACAGTCATTTGCTGCTGCTGCTGCTGCTGCTGCTGCTTCTCGAACTGGATGCATTAGGAAGCTGC

[0293] Underline shows the polymorphic repeat sequence.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:197 (2) INFORMATION FOR SEQ ID NO: Clone 22 upstream primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 1: CAAGTTTATG TTACTGCCAG GG 22 (2) INFORMATION FOR SEQ ID NO:Clone 22 downstream primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCAGCTTCCTAATGCATCCA G 21 (2) INFORMATION FOR SEQ ID NO: Clone 22 isoform 1,unspliced protein (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 306 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:mat_peptide (B) LOCATION: 1...306 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Pro Glu Ala Gly Phe Gln Ala Thr Asn Ala Phe Thr Glu Cys Lys 1 510 15 Phe Thr Cys Thr Ser Gly Lys Cys Leu Tyr Leu Gly Ser Leu Val Cys 2025 30 Asn Gln Gln Asn Asp Cys Gly Asp Asn Ser Asp Glu Glu Asn Cys Leu 3540 45 Leu Val Thr Glu His Pro Pro Pro Gly Ile Phe Asn Ser Glu Leu Glu 5055 60 Phe Ala Gln Ile Ile Ile Ile Val Val Val Val Thr Val Met Val Val 6570 75 80 Val Ile Val Cys Leu Leu Asn His Tyr Lys Val Ser Thr Arg Ser Phe85 90 95 Ile Asn Arg Pro Asn Gln Ser Arg Arg Arg Glu Asp Gly Leu Pro Gln100 105 110 Glu Gly Cys Leu Trp Pro Ser Asp Ser Ala Ala Pro Arg Leu GlyAla 115 120 125 Ser Glu Ile Met His Ala Pro Arg Ser Arg Asp Arg Phe ThrAla Pro 130 135 140 Ser Phe Ile Gln Arg Asp Arg Phe Ser Arg Phe Gln ProThr Tyr Pro 145 150 155 160 Tyr Val Gln His Glu Ile Asp Leu Pro Pro ThrIle Ser Leu Ser Asp 165 170 175 Gly Glu Glu Pro Pro Pro Tyr Gln Gly ProCys Thr Leu Gln Leu Arg 180 185 190 Asp Pro Glu Gln Gln Met Glu Leu AsnArg Glu Ser Val Arg Ala Pro 195 200 205 Pro Asn Arg Thr Ile Phe Asp SerAsp Leu Ile Asp Ile Ala Met Tyr 210 215 220 Ser Gly Gly Pro Cys Pro ProSer Ser Asn Ser Gly Ile Ser Ala Ser 225 230 235 240 Thr Cys Ser Ser AsnGly Arg Met Glu Gly Pro Pro Pro Thr Tyr Ser 245 250 255 Glu Val Met GlyHis His Pro Gly Ala Ser Phe Leu His His Gln Arg 260 265 270 Ser Asn AlaHis Arg Gly Ser Arg Leu Gln Phe Gln Gln Asn Asn Ala 275 280 285 Glu SerThr Ile Val Pro Ile Lys Gly Lys Asp Arg Lys Pro Gly Asn 290 295 300 LeuVal 305 (2) INFORMATION FOR SEQ ID NO: Clone 22 isoform 2 alternativelyspliced protein (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:mat_peptide (B) LOCATION: 1...288 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Pro Glu Ala Gly Phe Gln Ala Thr Asn Ala Phe Thr Glu Cys Lys 1 510 15 Phe Thr Cys Thr Ser Gly Lys Cys Leu Tyr Leu Gly Ser Leu Val Cys 2025 30 Asn Gln Gln Asn Asp Cys Gly Asp Asn Ser Asp Glu Glu Asn Cys Leu 3540 45 Leu Val Thr Glu His Pro Pro Pro Gly Ile Phe Asn Ser Glu Leu Glu 5055 60 Phe Ala Gln Ile Ile Ile Ile Val Val Val Val Thr Val Met Val Val 6570 75 80 Val Ile Val Cys Leu Leu Asn His Tyr Lys Val Ser Thr Arg Ser Phe85 90 95 Ile Asn Arg Pro Asn Gln Ser Arg Arg Arg Glu Asp Gly Leu Pro Gln100 105 110 Ile Met His Ala Pro Arg Ser Arg Asp Arg Phe Thr Ala Pro SerPhe 115 120 125 Ile Gln Arg Asp Arg Phe Ser Arg Phe Gln Pro Thr Tyr ProTyr Val 130 135 140 Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu SerAsp Gly Glu 145 150 155 160 Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr LeuGln Leu Arg Asp Pro 165 170 175 Glu Gln Gln Met Glu Leu Asn Arg Glu SerVal Arg Ala Pro Pro Asn 180 185 190 Arg Thr Ile Phe Asp Ser Asp Leu IleAsp Ile Ala Met Tyr Ser Gly 195 200 205 Gly Pro Cys Pro Pro Ser Ser AsnSer Gly Ile Ser Ala Ser Thr Cys 210 215 220 Ser Ser Asn Gly Arg Met GluGly Pro Pro Pro Thr Tyr Ser Glu Val 225 230 235 240 Met Gly His His ProGly Ala Ser Phe Leu His His Gln Arg Ser Asn 245 250 255 Ala His Arg GlySer Arg Leu Gln Phe Gln Gln Asn Asn Ala Glu Ser 260 265 270 Thr Ile ValPro Ile Lys Gly Lys Asp Arg Lys Pro Gly Asn Leu Val 275 280 285 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal(ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...18 (D) OTHERINFORMATION: alternatively spliced portion lacking from isoform 2 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5: Glu Gly Cys Leu Trp Pro Ser Asp SerAla Ala Pro Arg Leu Gly Ala 1 5 10 15 Ser Glu (2) INFORMATION FOR SEQ IDNO: Clone 22 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8065 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...8065 (ix) FEATURE: (A) NAME/KEY: misc_feature (B)LOCATION: 452...505 (D) OTHER INFORMATION: alternatively spliced portion(ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 5595...5685 (D)OTHER INFORMATION: amplified region for genotyping (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: CCCAGCAGAG CGATGGACTT GGACAGGCTA AGATGGAAGTGACCTGAGCC TCGCCCGGCG 60 GCTTCCTCGA CGGGACAGCG CAAGAGTTGG AGCACAGGCTTGTCCGGGGA GCAGTATGCC 120 GGAAGCTGGT TTTCAGGCCA CAAATGCTTT CACAGAGTGCAAATTCACCT GCACCAGTGG 180 TAAATGCTTG TATCTTGGTT CGCTGGTCTG TAACCAACAGAACGACTGTG GGGACAACAG 240 TGACGAAGAG AACTGTCTCC TGGTGACCGA GCACCCGCCTCCGGGCATCT TCAACTCGGA 300 GCTGGAGTTC GCCCAAATCA TCATCATCGT CGTGGTGGTCACGGTGATGG TGGTGGTCAT 360 CGTCTGCCTG CTGAACCACT ACAAAGTCTC CACGCGGTCCTTCATCAACC GCCCGAACCA 420 GAGCCGGAGG CGGGAGGACG GGCTGCCGCA GGAAGGGTGCCTGTGGCCTT CAGACAGCGC 480 CGCACCGCGG CTGGGCGCCT CGGAGATCAT GCATGCCCCGCGGTCCAGGG ACAGGTTCAC 540 AGCGCCGTCC TTCATCCAGA GGGATCGCTT CAGCCGCTTCCAGCCCACCT ACCCCTATGT 600 GCAGCACGAG ATTGATCTTC CTCCCACCAT CTCCCTGTCCGACGGTGAAG AGCCACCTCC 660 TTACCAGGGG CCCTGCACCC TGCAGCTCCG GGACCCTGAACAGCAGATGG AACTCAACCG 720 AGAGTCCGTG AGGGCCCCAC CCAACCGAAC CATATTTGACAGTGATTTAA TAGACATTGC 780 TATGTATAGC GGGGGTCCAT GCCCACCCAG CAGCAACTCGGGCATCAGTG CAAGCACCTG 840 CAGCAGTAAC GGGAGGATGG AGGGGCCACC CCCCACATACAGCGAGGTGA TGGGCCACCA 900 CCCAGGCGCC TCTTTCCTCC ATCACCAGCG CAGCAACGCACACAGGGGCA GCAGACTGCA 960 GTTTCAGCAG AACAATGCAG AGAGCACAAT AGTACCCATCAAAGGCAAAG ATAGGAAGCC 1020 TGGGAACCTG GTCTGATTCC TTCCAACGTG CACTTCAGCTGGAGAAAGAA ACCAAGAAGG 1080 GAAGCGGCCG CTGGGCCCCT CCTGCGCACA GTGTTGTTCAGTTTCACATG GTACAAATAA 1140 GTAAAACCAA ATGAGCAAAC ACGGTCTTTG TTTCTGATTCCTTTTAGGGG AATTGCATGC 1200 AAACTAGACT GAAATGATAC AAACTTCCAT CTGGTCTGACCGCAAACAGT GTTTATTTGG 1260 GGACAGGGGT TGGGATGGGG GTGTGGGCAG GGGAAAACAGAGAACGGGAT GCTTTGAAGA 1320 TACCATGAAA TAAAACCCAC AGAGGTATTT GATGTATTTAATTGTGAAAG GAGACTTTGC 1380 AGATAAATGA GGCCAGAATG GCATGTTTTA TAATTAACTGAATAAAGAAG GAAGCATTAT 1440 TATATATTAT TGTGGGGAAG AACCAGCCAG TTCGCTTTTTCTCCTAAGGT GTGGACTTTT 1500 ATTTTGTTTT AAAAATATGA ATCAAAATTC CTGTGTTGTGTGCCAAGGTA TAAAGTGGAG 1560 AAGTTAGATG AGTGCAAGGA GCTCCTTTGT GTTGTGATGATGTGTTTTAA AAGTTGCACT 1620 ATCTTAATGT TGAAAATATT TACAAGGGAA CTGTTTTACGTGAAGTTCTG TATGTTGTCT 1680 TTTCACCTGT GGATTGTAAT CAGGCCCAAG GAATATCCTGGAGTGGTCCC CAGAAGCATC 1740 CAAGAAAAGA TATTTGGGGA CGTAGCCTAA CATTTTACCAACTTACGTAA ATCAAAAAAG 1800 TCATTATTGT TGCAGGAGTT TGCATCAAAT AGCAGTGCATCGCTGAAGCT TTTGGAGACT 1860 TTTGGATGGA AGATAAGATA GGGAAGATTA AGTTCCAGCATTTCTGACTT GTTATTTTGA 1920 GTTACTCTGC TACTCTTAGG CTGCATAGTT TATGAGAAAATGAACACATG CATTTATGGA 1980 TCCAGTATCA TGCAGTGCTG CCCTCATCCT CCAGCAGTGCAATTTCTTCA GTAATTTAGA 2040 TTTTTTTCAC TATAGCATGA AATATATTCA AATACATACCTTATTTTATG CAATAAATTG 2100 TTTAAAATGC AAGGTGGTTA TTCTGCATAC TGTTGAAATATGTGACTCCT CAGTATATTC 2160 CCATTGCCTC TCCCCCTTTC CTCGACAGCT TAGTTCAGTTCTGCAGGGCT GCTCAGTTCA 2220 CAGGAGGCTC CCAGCAGCCA CCCCACATCC AGCCTACACAGAACTTTCGT GTGGGAGTGG 2280 TGTGGGTGGT GGTTTTCTTA TGCTTTGGAA GCCCCTAGAAATAATGACGG AAGAATGCCA 2340 TGTTGCTGAT CGTGGTAATA AGCCATTGTG GGTTATTGTATGTCACTAGT ATTAGCATAG 2400 CATTCTTAAA GGAATGCAGT GTTCAAAACC TACCCAAATTCCCCGCAGGA TTTTACCAAA 2460 CCCTTCCCCA GGCCAGTTTT GTACTGAAGG CAAGAACTGGACAGTCAGAG AACAGTGGAG 2520 GGGGCAAGTG ACTGAAGAGC ACCGGGTAAA AAGCACAACATGCAGTTAAA ATGCAAACTA 2580 GAAAACTAAT TTTAAATATT GTTAGTTTTA ATATTTCCTGATATTTACAA ATATTCATTC 2640 TTATATACAA TGAAAAAAAT AACTTTCTTC TGCAGATGTAAGCACTGGCT TTTATAAGAG 2700 CAGCAGCCAA CACGTTTAGC AGACACTGCG CGTGGAGAAGGGCTTATCTG CAGTACACTC 2760 TGCCATGTGG AGGGTGGGCC TCTGTGGCCT CTTCACATAACAAGATGAGC TGGAATGATG 2820 ATTCCATGAC TCCCACCTAT GCAGCCTTAA AGCCAAATCCGCGTGTGTGT GTTTGTGTCT 2880 GTCTGTGGGT CTCGAAGGTG ATCCGTCGGT GCGGTGGCTCTGTGCTGTAA CTGGAGAGAC 2940 TGTTCCAAAC CCCAAGAGTT GTCTGATCCT AGTCTGTTCCCTTCTGCTTC TTACCTCTGT 3000 AGATAGGTCA CTGGTTTTTG TTTGTTTGTT TTGAGGATTGGAATTTCCAT TACATTCATC 3060 CTTTGCACAC AGTAACATCC ACAGAACTAG TCCAACTCTTAAAAGGAGAG AGGAAAAACA 3120 CAGGCACCAG TTGTCAGCTC ATGCTTACAA CCTGTGTGGAAGTATATACA GTTGAGAGTC 3180 ACAGTGGAGG TTCTGAGACT GGATTCAGTC TTGTTCCAGTGACAGTTGGA AGGCCTCTGC 3240 TGGAGAGACA CCAGCTCTCA GGGCAGAGAT TGGCTTGGGGCCAGAAGGAC CCTCCCCAAC 3300 CCTGGAGACA CCCTGAAGGT TCACTGGCTC TCCAGATTAGCCTCTCTTCC TCTGTCAGGC 3360 AAAGATGAGG AGCCCGTGTT CCCATCGGGC CCTGCTGGCAGGGACTTGCA GTGGATTCTT 3420 GGTCAGGTGT GCCCACAGAT GCGGAGGCGA GGTGAGTGATTCCATCATTT CAGTTCTCAC 3480 CTGCAGTTTT GGTGAAGCAG GAGATGCACC CCACAGCTCTAGCTCTCAAA TGGCTTCACA 3540 GTCCTTACTT CTCTACCTGC CTCAAGAAGG GGCTCAGAGCAGAGACTTGT GAATTCCTTA 3600 GTAACTGTGA GTATATGAAT GTGTTGCACA TGTCCACAGTATTGGCGAGA TAATTACATA 3660 ATTCAGATAC CTTTAATCAT CTTTCAAGAA AGAGGCTCCTCCCATTCAAC CACCCTAGAG 3720 AACTGCCTTT GTTAAATAGT TATTTAAAGA CTCATACATATCAAACCATG ACTTTGAAAG 3780 GTCTTCGAGG CTGGGGCTCT GTAATGAATT AGTTTAAAAGCCAAGGTCAT AACATGAATT 3840 GATGGTCAAT TTCCCTTCAG CAGAAGGAAA AGGTGATTTAGATCAGTAGC TCTTTTGAAG 3900 GTTGTGGCTG ACCTGTTCAT ACCGTGTCGC CTCATGGCTAGTGTGGCGTT GAAAGAGTAG 3960 CGACTGGGAA GATACAACTT ACACAGTGGG GCCTATTGTTCTTTCAAGAA CCCTTTTTTT 4020 AGCTTATAGA ACCCATGGGT CCAGTTTAGT AACGAGTGATTTAGGCAATC AATGATAGGT 4080 TTATAATCTT AGATTATTCC AGCAAAGTGT GGATTGCATTGTTAGGAAGA ACATTTGGTG 4140 GGAATGAACA CTCCTGGGCA TACCGCTGAC TTTTGTCCCTTGTTCCCGGT GTAGGAGACC 4200 CAAGGCATCT TGAATCCCAT CTATAAGAAC ACAATCTTCCAGCATACGTT TGCTTTTTCA 4260 GAAACTCTAG CATTCTCTTT AAATACTGAC GCAATCCTTAATGGAAAAGA GATTTCATGA 4320 AGCAAATTAT GTATTTCAAT AGTTCTTCTA TTTTTAGTGTCCAAAATTTA CTAATACAGA 4380 AGCTTGACAA GCATGTCCTC ACCCTCCCCA CCACATAAACACATGGACAC ACACCCAAGC 4440 CACAAGAAAT CCCAAGAGAG CAGAAGCGAA TTTTTAAAAGATTTATCGTG AGGACTGCAT 4500 TTCCATTCAC TAATTTTGGC TCAAACTTAT GAGGCAGGAAATAGGGGCCA ACAGTAAATG 4560 GGGGAGGCCT CCTGACACCA GCAGAGGAAT TTTGTACCCAGGCGAGGACT TCTTGAACTT 4620 CTGCGTATCT CCGTTTGATC TCTTTCACCT TTATTTCATCTTCATAAGAA TGAGAAAGGC 4680 TCAAAAGGAA GCACTTTTAG AAATCTTCTC TGACCTAGAAGAATCCATCC AAATCCCTGC 4740 CTTCCTCTCT GAACCAACAG TTCCCTTCTC TGACAGGGGGCCATCCTCTA TCTTCCATCC 4800 AGCGGCTCTT CCTTTTAGGA AGGCTCTGGT GCAGAGCACTTCAAATATGT CCTCAGGCCA 4860 GATACTGATT GCTAGTAGAG AGACACCCGG CACCCAGTCCGAAGCCCTCC CTCAAAGGAC 4920 CGGCTTATGG CGTTGGTCAC TGGCAGGCTC AGAGACATTCTACTGTGGGC GCAGGGAGCC 4980 CGGCCCCCCA TGCAGCCATG ACTGGATGCG CCCCCATCTCGGGGGCTTGC TGCACTGCTT 5040 GTTTATTGAA TTTTGCTACT TAGAATGGCA ACATTAACTTTGTGTACCAT TCATTTTTTA 5100 AAAATTTTCC AAAGCTCGGC AGTGTATGAA AGAAAAAACTGGGAAAGATA CTTGGTTTCT 5160 GTTAACTTTT GTGTTGCTTG CTTAAGTGAT TAAAGCCAGTGCTTGGAGCC AAGCCTTCAT 5220 GCCACGAACA TGCTCCACAG CCTGCCCTTT GCTCTCCTGCTCACACTGAC CAAGAATGCC 5280 GCGTGCTTGG CCTACTGAGG TGAAAGGACA ATTGAATGACAGGTGGGCAA AGGGAGAACT 5340 TCCCCTTCTT GGTGCGAGGA AAGTCACAAA TTTAAAAATGTTGCTTCCAG CCCAGATCCT 5400 AAATGCTAGT TCTCAGCAGC TGCGTGGCTT ACCGTTCGCCATTTCCACCA CCGCCAGCTG 5460 CCAGCACCGC TACAGATCAC AGAGATGTGA ACAGACAATGGAAAGCACTC TTAGCCTTGC 5520 AGTGGTCTAC ATTTTTTAGG AACCAATATT TCAGCATTCTTTATTACCCG GCACGCTGTG 5580 TCCTTTGCAG AGTTCAAGTT TATGTTACTG CCAGGGTCAGACAGTCATTT GCTGCTGCTG 5640 CTGCTGCTGC TGCTGCTTCT CGAACTGGAT GCATTAGGAAGCTGCTGTCT GAGTGTAGGA 5700 ATGTCTTGCT AAGAAAGCAA TGTCTTCCTT CATCCTTTTCTTTCTTCCCT CTGCGTGTCC 5760 TTGTTTTTGT GTAATGCGGG AGAGGGTTAG AGCTATAGAGATTATATATA CACTATCCGT 5820 GCACATTATA TATATGTAGA TATACCCCTA TCATGTCAGAGATCTGCATG TCAGTTTTTC 5880 AGCAACTAAG GTGCCTCATG TTCTGAGTTC AGCAGATATAGGAACCAAGC CGCCCCCTCC 5940 TGCACTTGAT GCTCCCACCT TTGTTGTGCC TCACTTAAAATGGTGCTTTT TTCAGTTGTC 6000 TGTCTTTTCT TATGTTTTTA TTTGTAAGGT GCTGTATATAAGTTGAATAT ATTATGCACA 6060 TATCCTACCC AATGGGTAGA ACAAAAAGTT GTTAATACTGTAATATAATG TATAGATGAT 6120 ACCAATTTTA ACAGAAATGG CATAGAATTT GTGAATGCCTATGTGCTTTG TCCTCTTTTG 6180 TAAGGAAATT TGCAAATGGA TGCATACAGA TTAAAGTCTATGTAGTTTAT TTTCCTATTA 6240 AATATCAATA TTATAACACA AGAGAAAGAA GTGTGAACAAACAAGCAACA GTTTATGACC 6300 AGCGTATATA TAGCAATGGA AAGTTGCATC TTTGCTGTGAAAACACTTTA AAGAAAATAC 6360 TTTTTAAAAA ATCCCACAGC TTTTTGGTTG CCACTAGACGCTTCTTATTT TAATCATTTT 6420 AGTAATGCTC AGCTGGACCA GTGTTAGTTA TATTTGAGTCAGAAAAATGT TGTTTTTCAA 6480 CTTGCTTTAT AATCTCCTGC ATCTATCTCC TGCTGTAGCATCAYGAAGGT GTCAGGCAAC 6540 AGTGAAAAGT GCACATTTTT GTTGTTGCAG AAACTGTGTCAGAGGAATAA GTAAATCAGC 6600 CTGCAGCAGA AGACTTTGTT CAGCTCCAGA GGCATCTGTGACCGTCTGTG TCCAAGTCTC 6660 TCTGTGCCTT TTTCTTTTAC AAACTGAAGC TGTGGAGCCAATGAAGTAAC AGTAGAGATT 6720 GTAGGGAAAG AATACCTCAG GAAAAACAAA TACACTTACAAGAAGACCCT GTTCTTAGAA 6780 AATGTGTTTA GTTATGGGTT AGCACTAGAA GAGACTTGGCTGTCAGCCAG CCAAGTGAAG 6840 GACCTCTCAT CCATTCCCAT TCATGTCCCA TCATAATACGGACMCAAAAA GCAAACTCGG 6900 TTTTGCCATC AGTTAGAAAT TACGTTTTGG ATTGTATATTGTTACATCTC TCTTCCAGCT 6960 TAGTTTTTAG TGTCTGATTG TGACCTCTGC ATTTATCTTCAAATACCCTA ATTTTAAAAC 7020 AAAAGAACAA GAAAAGTTTA TAACACCATG TTCACTAAAACCACGGTTGA ATCTTGGGTG 7080 TGGGCATCCT TTCGAGTGTT GTCCATAAGA GCAGTTCGTGGAATTTTGCC CATCTGACCC 7140 ATATTATCAG CTTATTCTGC CACCAGAGTA GAGTCTAATAAATTCCAAAG TTTTTATTTG 7200 CTCCATGGTG TATGTTCTGA CTTTGAAAAT GTCAGATTCTATAATCATAC CCCTAACATC 7260 CAGGAGACAA ATGACAGATT ATCTTTAAAC TGAAATTGACTCTACAATGC AACCCTTAAT 7320 GCTGAATGGA TTAAAAAAGT CAGCCCTTTT AGTATCTGTTTGAAAGGGCC GTAAAAAGTT 7380 GACACTTTTG TTGTTGTGGA TCCTGCGTGT CTAGACCCACGTGTTGTTTC CATCGTATAC 7440 TGTAGGGTGC ACCCCTTGGG ATTCATCATT AAGAACTGAGGCTCACTGTT GTCAGAAACA 7500 AAGCTCCCAC CCCCCAGGTT CAACCTTGTG GGAGAACTGTTGAGCATGAG AATGTTCTAG 7560 ACTCAGAGGT ACTAAAATTT GTTACCACAT CATTGCTTCCTTTCTACAGG ACGAATTGAG 7620 GCTTAAACTT TACTGTTAAT GATACTGGTT CATTTTAATGTGCTTGTTGG TATGTTGCTA 7680 TTTTTCATTT CATAGCTTTC AAAAATCATG CTAATTGTATACTTGTCTAN TTTAAGGCTA 7740 TTTTAAAATA TGTACAATAC TATTCACAGC ATTTAGTTCGTTTAATTTTT ATTATAAAGC 7800 AATCTACTAA AAAAGTACAA CTGTATTTGA ACTTTTCAATAGTTGTTTGT GAGCTATGAT 7860 AATCAAAAGT CATTAAAGTC TTTTTTAACA AACATTCGTGCTTACTTTTC AACATAATTC 7920 CCAGTTATAT ACAGAAAAAG ATTTCCACCT GTCACGTATCTGCCTCTTTT ACCTGAGCAA 7980 TGGTGTAGTT CTTANACCTA AGGTCTGTAA TTGCAATACTTTTAAAGAAA GAGTTGCTCT 8040 AAGTGCTGTT TGTTAGTTAT GAAAC 8065 (2)INFORMATION FOR SEQ ID NO: Clone 22 coding region (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 921 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1...921 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 7: ATGCCGGAAG CTGGTTTTCA GGCCACAAAT GCTTTCACAGAGTGCAAATT CACCTGCACC 60 AGTGGTAAAT GCTTGTATCT TGGTTCGCTG GTCTGTAACCAACAGAACGA CTGTGGGGAC 120 AACAGTGACG AAGAGAACTG TCTCCTGGTG ACCGAGCACCCGCCTCCGGG CATCTTCAAC 180 TCGGAGCTGG AGTTCGCCCA AATCATCATC ATCGTCGTGGTGGTCACGGT GATGGTGGTG 240 GTCATCGTCT GCCTGCTGAA CCACTACAAA GTCTCCACGCGGTCCTTCAT CAACCGCCCG 300 AACCAGAGCC GGAGGCGGGA GGACGGGCTG CCGCAGGAAGGGTGCCTGTG GCCTTCAGAC 360 AGCGCCGCAC CGCGGCTGGG CGCCTCGGAG ATCATGCATGCCCCGCGGTC CAGGGACAGG 420 TTCACAGCGC CGTCCTTCAT CCAGAGGGAT CGCTTCAGCCGCTTCCAGCC CACCTACCCC 480 TATGTGCAGC ACGAGATTGA TCTTCCTCCC ACCATCTCCCTGTCCGACGG TGAAGAGCCA 540 CCTCCTTACC AGGGGCCCTG CACCCTGCAG CTCCGGGACCCTGAACAGCA GATGGAACTC 600 AACCGAGAGT CCGTGAGGGC CCCACCCAAC CGAACCATATTTGACAGTGA TTTAATAGAC 660 ATTGCTATGT ATAGCGGGGG TCCATGCCCA CCCAGCAGCAACTCGGGCAT CAGTGCAAGC 720 ACCTGCAGCA GTAACGGGAG GATGGAGGGG CCACCCCCCACATACAGCGA GGTGATGGGC 780 CACCACCCAG GCGCCTCTTT CCTCCATCAC CAGCGCAGCAACGCACACAG GGGCAGCAGA 840 CTGCAGTTTC AGCAGAACAA TGCAGAGAGC ACAATAGTACCCATCAAAGG CAAAGATAGG 900 AAGCCTGGGA ACCTGGTCTG A 921 (2) INFORMATIONFOR SEQ ID NO: Clone 22 isoform 2 alternatively spliced coding region(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 867 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1...867 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGCCGGAAG CTGGTTTTCA GGCCACAAATGCTTTCACAG AGTGCAAATT CACCTGCACC 60 AGTGGTAAAT GCTTGTATCT TGGTTCGCTGGTCTGTAACC AACAGAACGA CTGTGGGGAC 120 AACAGTGACG AAGAGAACTG TCTCCTGGTGACCGAGCACC CGCCTCCGGG CATCTTCAAC 180 TCGGAGCTGG AGTTCGCCCA AATCATCATCATCGTCGTGG TGGTCACGGT GATGGTGGTG 240 GTCATCGTCT GCCTGCTGAA CCACTACAAAGTCTCCACGC GGTCCTTCAT CAACCGCCCG 300 AACCAGAGCC GGAGGCGGGA GGACGGGCTGCCGCAGATCA TGCATGCCCC GCGGTCCAGG 360 GACAGGTTCA CAGCGCCGTC CTTCATCCAGAGGGATCGCT TCAGCCGCTT CCAGCCCACC 420 TACCCCTATG TGCAGCACGA GATTGATCTTCCTCCCACCA TCTCCCTGTC CGACGGTGAA 480 GAGCCACCTC CTTACCAGGG GCCCTGCACCCTGCAGCTCC GGGACCCTGA ACAGCAGATG 540 GAACTCAACC GAGAGTCCGT GAGGGCCCCACCCAACCGAA CCATATTTGA CAGTGATTTA 600 ATAGACATTG CTATGTATAG CGGGGGTCCATGCCCACCCA GCAGCAACTC GGGCATCAGT 660 GCAAGCACCT GCAGCAGTAA CGGGAGGATGGAGGGGCCAC CCCCCACATA CAGCGAGGTG 720 ATGGGCCACC ACCCAGGCGC CTCTTTCCTCCATCACCAGC GCAGCAACGC ACACAGGGGC 780 AGCAGACTGC AGTTTCAGCA GAACAATGCAGAGAGCACAA TAGTACCCAT CAAAGGCAAA 840 GATAGGAAGC CTGGGAACCT GGTCTGA 867(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: primer A (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGCCGGAAG CTGGTTTTCA GG 22 (2)INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...23 (D) OTHER INFORMATION: primer B (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 10: TCCAGCTGAA GTGCACGTTG GCT 23 (2) INFORMATIONFOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...14 (D) OTHER INFORMATION: Motif A (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 11: Trp Xaa Ile Asp Pro Ile Asp Gly Thr Xaa XaaPhe Xaa His 1 5 10 (2) INFORMATION FOR SEQ ID NO: Clone 22 5′untranslated region (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY:5′UTR (B) LOCATION: 1...44 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:TGCGAGAGCC GGGCAGGTGG GCCGCGGATG CTCCCAGAGG CCGG 44 (2) INFORMATION FORSEQ ID NO: Clone 22 5′ untranslated region (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)FEATURE: (A) NAME/KEY: 5′UTR (B) LOCATION: 1...491 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 13: ATTTGCAGTA GAGGTGGATA GAGATGGTGA GCAGCATTGACTCTCAAAAA TAGGGTCCTA 60 TGGCTGGTAA GGAGGTTGGT GCCTTCTCGA AGGGCTAGTGCTGGGAAGCT TCCTTTTAAA 120 AACGGCCCTT TCTGCCGGTT TGGCTAGCCA AGAATGGCATCCTCCTCTCT GTATCTTCCC 180 TGGAGCTTCA GGACTGAGTA TTGAATGACA GAGAAGGTTCTGCAAAGTCT GCACAGGGAG 240 ACTGCCATTG CATCAAGTCA TGTCTGCATT CTGTATATGCGGTTCAAGCT CTACGTTCGT 300 GACATCAAAC CTCCTGTTGG GCCATTTCCG AGAACTCCCATCAGTTTCTG TATAGTGTAA 360 AAGTTTCAGA GGCGGAGGAC AGAGAGCTGC GGCTGGGACAAGGAGCACCC GCGTGCAGGT 420 GCGACCCTGC AGGATGCTGG CAGCGGCGTG GCCAGGGGCGCCCGTGTTCT GAGGGCCTGA 480 GGGCCAGCCC C 491 (2) INFORMATION FOR SEQ IDNO: Clone 22 allele 1 polymorphic marker (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...94 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 14: CAAGTTTATG TTACTGCCAG GGTCAGACAG TCATTTGCTG CTGCTGCTGCTGCTGCTGCT 60 GCTGCTTCTC GAACTGGATG CATTAGGAAG CTGC 94 (2) INFORMATIONFOR SEQ ID NO: Clone 22 allele 2 polymorphic marker (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 91 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...91 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 15: CAAGTTTATG TTACTGCCAG GGTCAGACAGTCATTTGCTG CTGCTGCTGC TGCTGCTGCT 60 GCTTCTCGAA CTGGATGCAT TAGGAAGCTG C91 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1447 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 142...1008 (D) OTHER INFORMATION: IMP.18pmyo-inositol monophosphatase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:GTGGGACGGG CGGCGGACTA GGCACAGAGC TGCGGGAGCA GGCACAGGGA GTGTGGAGCC 60TGGCGGCGGG ACGGCGGGAT CCGGTGGGAG CCGGAGTCCC GCCGAGGGGG GCTGGAGGTG 120GAGGGGCCCG GCGAGGCCGC G ATG AAG CCG AGC GGC GAG GAC CAG GCG GCG 171 MetLys Pro Ser Gly Glu Asp Gln Ala Ala 1 5 10 CTG GCG GCC GGC CCC TGG GAGGAG TGC TTC CAG GCG GCC GTG CAG CTG 219 Leu Ala Ala Gly Pro Trp Glu GluCys Phe Gln Ala Ala Val Gln Leu 15 20 25 GCG CTG CGG GCA GGA CAG ATC ATCAGA AAA GCC CTT ACT GAG GAA AAA 267 Ala Leu Arg Ala Gly Gln Ile Ile ArgLys Ala Leu Thr Glu Glu Lys 30 35 40 CGT GTC TCA ACA AAA ACA TCA GCT GCAGAT CTT GTG ACA GAA ACA GAT 315 Arg Val Ser Thr Lys Thr Ser Ala Ala AspLeu Val Thr Glu Thr Asp 45 50 55 CAC CTT GTG GAA GAT TTA ATT ATT TCT GAGTTG CGA GAG AGG TTT CCT 363 His Leu Val Glu Asp Leu Ile Ile Ser Glu LeuArg Glu Arg Phe Pro 60 65 70 TCA CAC AGG TTC ATT GCA GAA GAG GCC GCG GCTTCT GGG GCC AAG TGT 411 Ser His Arg Phe Ile Ala Glu Glu Ala Ala Ala SerGly Ala Lys Cys 75 80 85 90 GTG CTC ACC CAC AGC CCG ACG TGG ATC ATC GACCCC ATC GAC GGC ACC 459 Val Leu Thr His Ser Pro Thr Trp Ile Ile Asp ProIle Asp Gly Thr 95 100 105 TGC AAT TTT GTG CAC AGA TTC CCG ACT GTG GCGGTT AGC ATT GGA TTT 507 Cys Asn Phe Val His Arg Phe Pro Thr Val Ala ValSer Ile Gly Phe 110 115 120 GCT GTT CGA CAA GAG CTT GAA TTC GGA GTG ATTTAC CAC TGC ACA GAG 555 Ala Val Arg Gln Glu Leu Glu Phe Gly Val Ile TyrHis Cys Thr Glu 125 130 135 GAG CGG CTG TAC ACG GGC CGG CGG GGT CGG GGCGCC TTC TGC AAT GGC 603 Glu Arg Leu Tyr Thr Gly Arg Arg Gly Arg Gly AlaPhe Cys Asn Gly 140 145 150 CAG CGG CTC CGG GTC TCC GGG GAG ACA GAT CTCTCA AAG GCC TTG GTT 651 Gln Arg Leu Arg Val Ser Gly Glu Thr Asp Leu SerLys Ala Leu Val 155 160 165 170 CTG ACA GAA ATT GGC CCC AAA CGT GAC CCTGCG ACC CTG AAG CTG TTC 699 Leu Thr Glu Ile Gly Pro Lys Arg Asp Pro AlaThr Leu Lys Leu Phe 175 180 185 CTG AGT AAC ATG GAG CGG CTG CTG CAT GCCAAG GCG CAT GGG GTC CGA 747 Leu Ser Asn Met Glu Arg Leu Leu His Ala LysAla His Gly Val Arg 190 195 200 GTG ATT GGA AGC TCC ACA TTG GCA CTC TGCCAC CTG GCC TCA GGG GCC 795 Val Ile Gly Ser Ser Thr Leu Ala Leu Cys HisLeu Ala Ser Gly Ala 205 210 215 GCG GAT GCC TAT TAC CAG TTT GGC CTG CACTGC TGG GAT CTG GCG GCT 843 Ala Asp Ala Tyr Tyr Gln Phe Gly Leu His CysTrp Asp Leu Ala Ala 220 225 230 GCC ACA GTC ATC ATC AGA GAA GCA GGC GGCATC GTG ATA GAC ACT TCG 891 Ala Thr Val Ile Ile Arg Glu Ala Gly Gly IleVal Ile Asp Thr Ser 235 240 245 250 GGT GGA CCC CTC GAC CTC ATG GCT TGCAGA GTG GTT GCG GCC AGC ACC 939 Gly Gly Pro Leu Asp Leu Met Ala Cys ArgVal Val Ala Ala Ser Thr 255 260 265 CGG GAG ATG GCG ATG CTC ATA GCT CAGGCC TTA CAG ACC ATT AAC TAT 987 Arg Glu Met Ala Met Leu Ile Ala Gln AlaLeu Gln Thr Ile Asn Tyr 270 275 280 GGG CGG GAT GAT GAG AAG TGACTGCGGCTGAGGCAAAG CTGCTCCCAA GGCCTCCC 1043 Gly Arg Asp Asp Glu Lys 285TGGGCTGCTG TGGGCTCCTG GGGAGGTGGC CCTCGTGGCC CACGCTCCAT GCCAGTGCT 1103CACGCTCTGC TCCTGGCTAC CCCAGAGGGA GTTGTCACGC TACAGTGAGT GGCTGGCTT 1163TTAAATCGAC GTCTCTCTCA CCAGGATTTG GTGTTTAGCT GTTTCTCTCT TTAATCTAC 1223GTAGCCTTTT TCAGGTTAGT ACGTGTTCTT CTGTCAGGGC CAAAACTCAA ATCTCCTTG 1283AAATACGTAT TGATAATCCA ATCTTGATTT TTCCCCCCAG AATATAAATC TCAGGTATA 1343AGGCTTTAGA ACTGCTGATA AAGCGGATCG TTCTCAGGCC CTCCCCCCGG AGTACTTAG 1403AATGCAATAA ATCAAAATAA TGGGCAAAAA AAAAAAAAAA AAAA 1447 (2) INFORMATIONFOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY:linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:mat_peptide (B) LOCATION: 1...288 (D) OTHER INFORMATION: IMP.18pmyo-inositol monophosphatase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:Met Lys Pro Ser Gly Glu Asp Gln Ala Ala Leu Ala Ala Gly Pro Trp 1 5 1015 Glu Glu Cys Phe Gln Ala Ala Val Gln Leu Ala Leu Arg Ala Gly Gln 20 2530 Ile Ile Arg Lys Ala Leu Thr Glu Glu Lys Arg Val Ser Thr Lys Thr 35 4045 Ser Ala Ala Asp Leu Val Thr Glu Thr Asp His Leu Val Glu Asp Leu 50 5560 Ile Ile Ser Glu Leu Arg Glu Arg Phe Pro Ser His Arg Phe Ile Ala 65 7075 80 Glu Glu Ala Ala Ala Ser Gly Ala Lys Cys Val Leu Thr His Ser Pro 8590 95 Thr Trp Ile Ile Asp Pro Ile Asp Gly Thr Cys Asn Phe Val His Arg100 105 110 Phe Pro Thr Val Ala Val Ser Ile Gly Phe Ala Val Arg Gln GluLeu 115 120 125 Glu Phe Gly Val Ile Tyr His Cys Thr Glu Glu Arg Leu TyrThr Gly 130 135 140 Arg Arg Gly Arg Gly Ala Phe Cys Asn Gly Gln Arg LeuArg Val Ser 145 150 155 160 Gly Glu Thr Asp Leu Ser Lys Ala Leu Val LeuThr Glu Ile Gly Pro 165 170 175 Lys Arg Asp Pro Ala Thr Leu Lys Leu PheLeu Ser Asn Met Glu Arg 180 185 190 Leu Leu His Ala Lys Ala His Gly ValArg Val Ile Gly Ser Ser Thr 195 200 205 Leu Ala Leu Cys His Leu Ala SerGly Ala Ala Asp Ala Tyr Tyr Gln 210 215 220 Phe Gly Leu His Cys Trp AspLeu Ala Ala Ala Thr Val Ile Ile Arg 225 230 235 240 Glu Ala Gly Gly IleVal Ile Asp Thr Ser Gly Gly Pro Leu Asp Leu 245 250 255 Met Ala Cys ArgVal Val Ala Ala Ser Thr Arg Glu Met Ala Met Leu 260 265 270 Ile Ala GlnAla Leu Gln Thr Ile Asn Tyr Gly Arg Asp Asp Glu Lys 275 280 285 (2)INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...21 (D) OTHER INFORMATION: forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 18: ATGAAGCCGA GCGGCGAGGA C 21 (2) INFORMATIONFOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...22 (D) OTHER INFORMATION: reverse primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: CTTCTCATCA TCCCGCCCAT AG 22 (2) INFORMATIONFOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...22 (D) OTHER INFORMATION: forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 20: CTCGACCTCA TGGCTTGCAG AG 22 (2) INFORMATIONFOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...21 (D) OTHER INFORMATION: reverse primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 21: CTGAGAACGA TCCGCTTTAT C 21 (2) INFORMATIONFOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...21 (D) OTHER INFORMATION: forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 22: GTGTGTGCTC ACCCCGACTG T 21 (2) INFORMATIONFOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...21 (D) OTHER INFORMATION: reverse primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 23: CCCGAAGTGT CTATCACGAT G 21 (2) INFORMATIONFOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...29 (D) OTHER INFORMATION: clone #39740 specific primer p1(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ACGTCGGGCT GTGGGTGAGCACACACTTG 29 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 285 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 1...285 (D) OTHERINFORMATION: Xenopus IMP (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: MetGlu Asp Arg Trp Gln Glu Cys Met Asp Phe Leu Ala Val Ser Ile 1 5 10 15Ala Arg Lys Ala Gly Ser Val Val Cys Ala Ala Leu Lys Glu Asp Val 20 25 30Ser Ile Met Val Lys Thr Ser Leu Ala Pro Ala Asp Leu Val Thr Ala 35 40 45Thr Asp Gln Lys Val Glu Glu Met Ile Ile Ser Ser Ile Lys Glu Lys 50 55 60Tyr Pro Ser His Ser Phe Ile Gly Glu Glu Ser Val Ala Ala Gly Ala 65 70 7580 Gly Ser Thr Leu Thr Asp Asn Pro Thr Trp Ile Ile Asp Pro Ile Asp 85 9095 Gly Thr Thr Asn Phe Val His Arg Phe Pro Phe Val Ala Val Ser Ile 100105 110 Gly Phe Ala Val His Lys Gln Val Glu Phe Gly Val Val Tyr Ser Cys115 120 125 Val Glu Asp Lys Met Tyr Thr Gly Arg Lys Gly Lys Gly Ser PheCys 130 135 140 Asn Gly Gln Lys Leu Gln Val Ser Gly Gln Lys Asp Ile ThrLys Ser 145 150 155 160 Met Ile Ile Thr Glu Leu Gly Ser Asn Arg Asn ProGlu Phe Ile Lys 165 170 175 Thr Val Ser Leu Ser Asn Met Glu Arg Leu LeuCys Ile Pro Ile His 180 185 190 Gly Ile Arg Ala Val Gly Thr Ala Ala ValAsn Met Cys Leu Val Ala 195 200 205 Thr Gly Gly Ala Asp Ala Tyr Tyr GluMet Gly Leu His Cys Trp Asp 210 215 220 Met Ala Ala Ala Ser Val Ile ValThr Glu Ala Gly Gly Thr Ile Leu 225 230 235 240 Asp Ala Thr Gly Gly LeuPhe Asp Leu Met Ser Cys Arg Ile Ile Ser 245 250 255 Ala Ser Ser Arg GluIle Ala Glu Arg Ile Ala Lys Glu Leu Gln Ile 260 265 270 Ile Pro Leu GluArg Asp Asp Gly Lys Ser Thr Asn Ser 275 280 285 (2) INFORMATION FOR SEQID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 277 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: mat_peptide (B)LOCATION: 1...277 (D) OTHER INFORMATION: rat IMP (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 26: Met Ala Asp Pro Trp Gln Glu Cys Met Asp TyrAla Val Thr Leu Ala 1 5 10 15 Arg Gln Ala Gly Glu Val Val Cys Glu AlaIle Lys Asn Glu Met Asn 20 25 30 Val Met Leu Lys Ser Ser Pro Val Asp LeuVal Thr Ala Thr Asp Gln 35 40 45 Lys Val Glu Lys Met Leu Ile Ser Ser IleLys Glu Lys Tyr Pro Ser 50 55 60 His Ser Phe Ile Gly Glu Glu Ser Val AlaAla Gly Glu Lys Ser Ile 65 70 75 80 Leu Thr Asp Asn Pro Thr Trp Ile IleAsp Pro Ile Asp Gly Thr Thr 85 90 95 Asn Phe Val His Arg Phe Pro Phe ValAla Val Ser Ile Gly Phe Ala 100 105 110 Val Asn Lys Lys Ile Glu Phe GlyVal Val Tyr Ser Cys Val Glu Gly 115 120 125 Lys Met Tyr Thr Ala Arg LysGly Lys Gly Ala Phe Cys Asn Gly Gln 130 135 140 Lys Leu Gln Val Ser GlnGln Glu Asp Ile Thr Lys Ser Leu Leu Val 145 150 155 160 Thr Glu Leu GlySer Ser Arg Thr Pro Glu Thr Val Arg Met Val Leu 165 170 175 Ser Asn MetGlu Lys Leu Phe Cys Ile Pro Val His Gly Ile Arg Ser 180 185 190 Val GlyThr Ala Ala Val Asn Met Cys Leu Val Ala Thr Gly Gly Ala 195 200 205 AspAla Tyr Tyr Glu Met Gly Ile His Cys Trp Asp Val Ala Gly Ala 210 215 220Gly Ile Ile Val Thr Glu Ala Gly Gly Val Leu Met Asp Val Thr Gly 225 230235 240 Gly Pro Phe Asp Leu Met Ser Arg Arg Val Ile Ala Ala Asn Asn Arg245 250 255 Ile Leu Ala Glu Arg Ile Ala Lys Glu Ile Gln Val Ile Pro LeuGln 260 265 270 Arg Asp Asp Glu Ser 275 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 277 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: mat_peptide (B)LOCATION: 1...277 (D) OTHER INFORMATION: bovine IMP (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 27: Met Ala Asp Pro Trp Gln Glu Cys Met Asp TyrAla Val Thr Leu Ala 1 5 10 15 Gly Gln Ala Gly Glu Val Val Arg Glu AlaLeu Lys Asn Glu Met Asn 20 25 30 Ile Met Val Lys Ser Ser Pro Ala Asp LeuVal Thr Ala Thr Asp Gln 35 40 45 Lys Val Glu Lys Met Leu Ile Thr Ser IleLys Glu Lys Tyr Pro Ser 50 55 60 His Ser Phe Ile Gly Glu Glu Ser Val AlaAla Gly Glu Lys Ser Ile 65 70 75 80 Leu Thr Asp Asn Pro Thr Trp Ile IleAsp Pro Ile Asp Gly Thr Thr 85 90 95 Asn Phe Val His Gly Phe Pro Phe ValAla Val Ser Ile Gly Phe Val 100 105 110 Val Asn Lys Lys Met Glu Phe GlyIle Val Tyr Ser Cys Leu Glu Asp 115 120 125 Lys Met Tyr Thr Gly Arg LysGly Lys Gly Ala Phe Cys Asn Gly Gln 130 135 140 Lys Leu Gln Val Ser HisGln Glu Asp Ile Thr Lys Ser Leu Leu Val 145 150 155 160 Thr Glu Leu GlySer Ser Arg Thr Pro Glu Thr Val Arg Ile Ile Leu 165 170 175 Ser Asn IleGlu Arg Leu Leu Cys Leu Pro Ile His Gly Ile Arg Gly 180 185 190 Val GlyThr Ala Ala Leu Asn Met Cys Leu Val Ala Ala Gly Ala Ala 195 200 205 AspAla Tyr Tyr Glu Met Gly Ile His Cys Trp Asp Val Ala Gly Ala 210 215 220Gly Ile Ile Val Thr Glu Ala Gly Gly Val Leu Leu Asp Val Thr Gly 225 230235 240 Gly Pro Phe Asp Leu Met Ser Arg Arg Val Ile Ala Ser Ser Asn Lys245 250 255 Thr Leu Ala Glu Arg Ile Ala Lys Glu Ile Gln Ile Ile Pro LeuGln 260 265 270 Arg Asp Asp Glu Asp 275 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 277 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: mat_peptide (B)LOCATION: 1...277 (D) OTHER INFORMATION: human IMP (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 28: Met Ala Asp Pro Trp Gln Glu Cys Met Asp TyrAla Val Thr Leu Ala 1 5 10 15 Arg Gln Ala Gly Glu Val Val Cys Glu AlaIle Lys Asn Glu Met Asn 20 25 30 Val Met Leu Lys Ser Ser Pro Val Asp LeuVal Thr Ala Thr Asp Gln 35 40 45 Lys Val Glu Lys Met Leu Ile Ser Ser IleLys Glu Lys Tyr Pro Ser 50 55 60 His Ser Phe Ile Gly Glu Glu Ser Val AlaAla Gly Glu Lys Ser Ile 65 70 75 80 Leu Thr Asp Asn Pro Thr Trp Ile IleAsp Pro Ile Asp Gly Thr Thr 85 90 95 Asn Phe Val His Arg Phe Pro Phe ValAla Val Ser Ile Gly Phe Ala 100 105 110 Val Asn Lys Lys Ile Glu Phe GlyVal Val Tyr Ser Cys Val Glu Gly 115 120 125 Lys Met Tyr Thr Ala Arg LysGly Lys Gly Ala Phe Cys Asn Gly Gln 130 135 140 Lys Leu Gln Val Ser GlnGln Glu Asp Ile Thr Lys Ser Leu Leu Val 145 150 155 160 Thr Glu Leu GlySer Ser Arg Thr Pro Glu Thr Val Arg Met Val Leu 165 170 175 Ser Asn MetGlu Lys Leu Phe Cys Ile Pro Val His Gly Ile Arg Ser 180 185 190 Val GlyThr Ala Ala Val Asn Met Cys Leu Val Ala Thr Gly Gly Ala 195 200 205 AspAla Tyr Tyr Glu Met Gly Ile His Cys Trp Asp Val Ala Gly Ala 210 215 220Gly Ile Ile Val Thr Glu Ala Gly Gly Val Leu Met Asp Val Thr Gly 225 230235 240 Gly Pro Phe Asp Leu Met Ser Arg Arg Val Ile Ala Ala Asn Asn Arg245 250 255 Ile Leu Ala Glu Arg Ile Ala Lys Glu Ile Gln Val Ile Pro LeuGln 260 265 270 Arg Asp Asp Glu Asp 275 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1215 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...1215(D) OTHER INFORMATION: IMP.18p promoter sequence (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 1...1026 (D) OTHER INFORMATION: 5′flanking region (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1027...1215 (D) OTHER INFORMATION: upstream portion of exon 1 (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1027 (D) OTHERINFORMATION: minor transcriptional start site (ix) FEATURE: (A)NAME/KEY: misc_feature (B) LOCATION: 1033 (D) OTHER INFORMATION: majorcap site (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1193...1195 (D) OTHER INFORMATION: translational initiation codon (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1193...1215 (D) OTHERINFORMATION: complementary sequence to IMP.18p-specific antisenseoligonucleotide primer p (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:GTCCAGCCGC TGTCCTCGCA GTGTTTGGGG TTAGCGGAGG GAGAGCTTGT TTGCGACCAA 60ACTTGCCGCG CGGGGCGGCC GCTTGCAGGA ACACTGCGGC CTTGTGCGCT CGGCGGTCTG 120AGCTCCTGCG AGGCCGAGAA CGACGCCTAG CGCCCAGCGG CCTCCGCGAA CAAAAAGCGC 180CCGGTCGGAA GGTGCTGGCC CAGCCGCCCC TGGCGCTCGA GCCCGAATCC GGCCACAGAC 240CATGAAGGGA CGCCCGGCAC CACGTGCGCG GGGATCCGCG GACGGGACGC TCCCCGGCAC 300CTCCGGGGCT GGGCCGGCAC CGCACGGTCC CACCCAGACA GTAGCTGCCC CGGGCCCCCA 360AAACAGCCGT TCCTAGCTCC TCCCTCCCAG TTTCTGCGGT GGCCCAAGCC GCCCTCCGCG 420CGCTTGACCC AGAACAGTAC GGAGTTCTGC ACGAGCCGGG GGTGGGGCCT GTCTCAGCGC 480GCGGCGGTGG GGCGGGGCTT GGACACGGGC CCGGCTCAAC TTGAGGGAGG CGGGGCTCGA 540GGCTCAGAGG AGTTGGAGCC CGCTCTGCGC GCTGCGGGAC GGGGCACGGC GGAGCAGGGT 600TGGGTCCGCC TCGAGCGGGG AGGGTGATGC TGCACCACAG GGGCGGGGCT GGAGGTAAAG 660CGCGGAGCGG AGAGGGACCA GGCTCGGCAC TGATTTGTGT TCAGGGCTAG CCCAGAGGGG 720CGGGGCCAGG TACGGGGCGC AGCCGGGAGC GGGAGGGGCG GTGCAGGACG GGGCCGGGCA 780CGGCGCGGGA AGAGGCCAGG AGCAGCAACG GGTGCGGGGC GGGGCCGGGA GCGTCAAGGG 840GCGGGGAAGA GGGGGGAATG GGCGGGGCCG AGCTCTGCGA GGGGCGAGGT GGGGAATGCA 900GAGCGGGGCC GGACGCGGGA GCAGGGAGCT GGGCGGGGAG CGGGGCGGGG AGCTGGGCTG 960GGCTCGGCAC GGGGCGGGGC GGAGGGTGGG GAGCGGAAAG CAGGACGCGC GGCTCCCGCG 1020GCCCGCTGGC TGCCCTTCCC GCCAGCGCAG GTGTGGGACG GGCGGCGGAC TAGGCACAGA 1080GCTGCGGGAG CAGGCACAGG GAGTGTGGAG CCTGGCGGCG GGACGGCGGG TCCGGTGGGA 1140GCCGGAGTCC CGCCGAGGGG GGCTGGAGGT GGAGGGGCCC GGCGAGGCCG CGATGAAGCC 1200GAGCGGCGAG GACCA 1215 (2) INFORMATION FOR SEQ ID NO: Clone 22 forwardprimer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 30: TACAAAAGAG GACAAAGCAC 20 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...17 (D) OTHER INFORMATION: D18S73 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 31: TGCCACTGCA ACAATGC 17 (2)INFORMATION FOR SEQ ID NO: Clone 22 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 32: GGTGCCTGTA TATAAGTTGA 20 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: 1...20 (D) OTHERINFORMATION: D18S73 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CCCAGCAATC AACCTTTAAG 20 (2) INFORMATION FOR SEQ ID NO: Clone 24forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTACAGAATA GAATACATGGCG 22 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...23 (D) OTHER INFORMATION: D18S869 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: TGTTTATTTG TTTGACTCAATGG 23 (2) INFORMATION FOR SEQ ID NO: Clone 24 reverse primer (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 36: GAGCTCTGAA CTGTATTCAG A 21 (2) INFORMATIONFOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...22 (D) OTHER INFORMATION: D18S869 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 37: GAGTGAATGC TGTACAAACA GC 22 (2)INFORMATION FOR SEQ ID NO: Clone 29 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...18 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 38: TCTCAGCTTA CTCAACCT 18 (2) INFORMATION FOR SEQ ID NO: 39:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...23 (D) OTHERINFORMATION: D18S996 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 39: GATGGAAAGC CATTTTATTT TTC 23 (2) INFORMATION FOR SEQ ID NO:Clone 29 reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: GATGAGGTGGAACAATCAC 19 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...25 (D) OTHER INFORMATION:D18S996 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:TCGTACTATG AAATTTTTAA GCCTT 25 (2) INFORMATION FOR SEQ ID NO: 42: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: GNAL (Clone 31) forward primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 42: GGTCTGTACA GTGTAATAAA CC 22 (2) INFORMATION FOR SEQ IDNO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20(D) OTHER INFORMATION: FB14A10 forward primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 43: CCTTCCCCTC TATTCTCAAA 20 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: GNAL (Clone 31) reverse primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 44: CTACTGCAAA ATGTGTCCTG TC 22 (2) INFORMATION FOR SEQ IDNO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20(D) OTHER INFORMATION: FB14A10 reverse primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 45: GAGCGAGACT GTCTCAAAAA 20 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: Clone 37 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 46: CACATTAGCC AGTCTGATAA AG 22 (2) INFORMATION FOR SEQ ID NO:GC32001 forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GAGTTGTGGGGGGGAATAGT 20 (2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION:Clone 37 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:AAGTTACACA CAGTAGCTGA 20 (2) INFORMATION FOR SEQ ID NO: GC32001 reverseprimer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...23 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 49: ATACGGAGGT TGAACTAGGA AGG 23 (2)INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...25 (D) OTHER INFORMATION: AFMa058yg5 forward primer(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: TAGATGCTAT ATTAGGCTGG GTCTC 25(2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: GP4B15 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: CGGTTCTGGA TTTATCAGTA20 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: AFMa058yg5reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GAACTTACAGCACTGGCTCT CC 22 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION:GP4B15 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:AGGGTTGCAA TGAGCTGAG 19 (2) INFORMATION FOR SEQ ID NO: AFMa152wg9forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: AAGAACAAAA GGTCACCTGTCA 22 (2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: IB-1114 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: GCCACACACA AATTTTTCTC20 (2) INFORMATION FOR SEQ ID NO: AFMa152wg9 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 56: TGTCTCACCT CTGCTCACTC AT 22 (2) INFORMATION FOR SEQ IDNO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20(D) OTHER INFORMATION: IB-1114 reverse primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 57: ACAGGGTGTA AGAGGAGAGG 20 (2) INFORMATION FOR SEQ ID NO:CHLC.GGAA16G02 forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:ATGGAAGGAA AAACAGAGGG 20 (2) INFORMATION FOR SEQ ID NO: NIB-1802 forwardprimer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 59: CTGATCACAT TTCATACAGC 20 (2)INFORMATION FOR SEQ ID NO: CHLC.GGAA16G02 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 60: GAACTCTTCA AGAGGGGAGC 20 (2) INFORMATION FOR SEQ ID NO:NIB-1802 reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: TGTATGTGGGCTTAACTGTT 20 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...25 (D) OTHER INFORMATION:D18S1114 forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:ATCAGTATAA TGATGGATGA ATCAC 25 (2) INFORMATION FOR SEQ ID NO: 63: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...24 (D) OTHERINFORMATION: SGC-31363 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 63: CTACTGGGAG GTAGGTAATC TCAG 24 (2) INFORMATION FOR SEQ ID NO: 64:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...17 (D) OTHERINFORMATION: D18S1114 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 64: TGAGGCAAGA GGGTCAC 17 (2) INFORMATION FOR SEQ ID NO: 65: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHERINFORMATION: SGC-31363 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 65: GCAAAACCAA CCACATCAAA 20 (2) INFORMATION FOR SEQ ID NO: 66: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...18 (D) OTHERINFORMATION: D18S1116 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 66: TCTGCCACTT TTTATGGG 18 (2) INFORMATION FOR SEQ ID NO: SGC34207forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: GATCCTGTTC TTTCAGCAGG20 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...25 (D) OTHER INFORMATION: D18S1116 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: CAATGTTTTA ACTTCTAGGACAAAT 25 (2) INFORMATION FOR SEQ ID NO: SGC34207 reverse primer (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 69: TTTAACCAGC TGGAGTGAAG G 21 (2) INFORMATIONFOR SEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...18 (D) OTHER INFORMATION: D18S1150 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 70: GGCACAGGAA ACGTGAAT 18 (2)INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...22 (D) OTHER INFORMATION: WI-11680 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 71: ACAGATACTT TTCCACGCAA CA 22 (2)INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...16 (D) OTHER INFORMATION: D18S1150 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 72: CACAAGGATG CCAGCC 16 (2)INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...21 (D) OTHER INFORMATION: WI-11680 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 73: AAAAAGATGT ACGGTCTGGC C 21 (2)INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...20 (D) OTHER INFORMATION: D18S1153 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 74: ATGGAGGCTC TGAGACCCTT 20 (2)INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...25 (D) OTHER INFORMATION: WI-13171 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 75: TTTTATTTGG ACAAGAGAAC TTGTG 25 (2)INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...19 (D) OTHER INFORMATION: D18S1153 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 76: CTTGCCTGAT GCCTGAAAT 19 (2)INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...20 (D) OTHER INFORMATION: WI-13171 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 77: ATGATCAGCT CTGAGGTGCA 20 (2)INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...20 (D) OTHER INFORMATION: D18S1158 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 78: GCATCTATGC AGTGCCAAAT 20 (2)INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...23 (D) OTHER INFORMATION: WI-18080 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 79: TGGCATAAAG TTTGCAAATA TCA 23 (2)INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...23 (D) OTHER INFORMATION: D18S1158 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 80: TCATTAGCAA CAAGGATCTC C 21 (2)INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: -(B) LOCATION: 1...25 (D) OTHER INFORMATION: WI-18080 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 81: ATACACCAAA GGAGAAGGAT TAACA 25 (2)INFORMATION FOR SEQ ID NO: D18S1228 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...24 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 82: AGACAGTTGA AAAGGACACA AATG 24 (2) INFORMATION FOR SEQ IDNO: 83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22(D) OTHER INFORMATION: D18S1066 forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 83: TGCTGTTGCC TCTCAGCATC TC 22 (2) INFORMATIONFOR SEQ ID NO: D18S1228 reverse primer (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TGGTGATGGG ACTTTTCAAA 20 (2) INFORMATION FOR SEQ ID NO: 85: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...23 (D) OTHERINFORMATION: D18S1066 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 85: CACCTTTCAA GTGCTTGGCA GTC 23 (2) INFORMATION FOR SEQ ID NO: 86:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: D18S378 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 86: AGCCTGGGTG ACAGAGCAA 19 (2) INFORMATION FOR SEQ ID NO: D18S1215forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: GTTTGCTGCA TCTCCCAATT20 (2) INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: D18S378 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: ACAGGGAAAG CTGGGGGAT 19(2) INFORMATION FOR SEQ ID NO: D18S1215 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 89: GTGCCCACAT TGTTGTGAAG 20 (2) INFORMATION FOR SEQ ID NO:90: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: D18S40 forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: CAAGATAGAT GCATTTTCCA GT 22 (2) INFORMATION FOR SEQ ID NO: D18S1299forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: TTTAAGCCTC AAGGGACCCT20 (2) INFORMATION FOR SEQ ID NO: 92: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: D18S40 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: CATCCAAAGG GTGAATGTGT20 (2) INFORMATION FOR SEQ ID NO: D18S1299 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 93: AGATTGAGGA CCAGGTGGTG 20 (2) INFORMATION FOR SEQ ID NO:94: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHERINFORMATION: D18S464 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 94: GCCAGACTTT GTGCCATTTC 20 (2) INFORMATION FOR SEQ ID NO: D18S1226forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: CTCTTAAGTT GAGTGAAGTGGAAGC 25 (2) INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...23 (D) OTHER INFORMATION:D18S464 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:TTTCCTGAAT CTCTTGTGGT TTG 23 (2) INFORMATION FOR SEQ ID NO: D18S1226reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: CGCAAAAGTC AGGAAAGAGG20 (2) INFORMATION FOR SEQ ID NO: D18S482 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 98: ATGAGTGAAT GCCAACTTCG 20 (2) INFORMATION FOR SEQ ID NO:SHGC-32282 forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: TTACGCATTTTGTATCAGAC TTACA 25 (2) INFORMATION FOR SEQ ID NO: D18S482 reverseprimer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 100: CCTGGCTGAC AGAGTGAGT 19 (2)INFORMATION FOR SEQ ID NO: SHGC-32282 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...25 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 101: GGTGGAGTAT CAGAAGTGAT TTTAG 25 (2) INFORMATION FOR SEQID NO: 102: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21(D) OTHER INFORMATION: D18S53 forward primer (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 102: GGTCACCTAC AACTTTGGAT G 21 (2) INFORMATION FOR SEQ IDNO: 103: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20(D) OTHER INFORMATION: D18S1315 forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 103: TGGACTTCTA CCCCCATCTG 20 (2) INFORMATIONFOR SEQ ID NO: 104: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...24 (D) OTHER INFORMATION: D18S53 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 104: TGCATGTAAA TATCAGAGTC TGTT 24 (2)INFORMATION FOR SEQ ID NO: 105: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: D18S1315 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: TTTGAAACCT GGACACTTTGG 21 (2) INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...16 (D) OTHER INFORMATION: D18S71 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: ACCCGCTCAA AAGCCT 16(2) INFORMATION FOR SEQ ID NO: 107: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: D18S843 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: GTCCTCATCT GTAAAACGGG20 (2) INFORMATION FOR SEQ ID NO: 108: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...25 (D) OTHER INFORMATION: D18S71 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: TTAATGGATT ATCAAGAGTGGTTCT 25 (2) INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...24 (D) OTHER INFORMATION:D18S843 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:CCACTAACTA GTTTGTGACT TTGG 24 (2) INFORMATION FOR SEQ ID NO: 110: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: Clone 1 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 110: AGGAGTGGTG TACATTTCT 19 (2) INFORMATION FOR SEQ ID NO: 111: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...23 (D) OTHERINFORMATION: Clone 1 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 111: ACCTGCAACA CATTAGAAAC 20 (2) INFORMATION FOR SEQ ID NO: Clone 2forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: GGTTTCTTCA AAATTTTATTAACAA 25 (2) INFORMATION FOR SEQ ID NO: Clone 2 reverse primer (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 113: TCCTCCACTC ATCTGTTTCT 20 (2) INFORMATIONFOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...19 (D) OTHER INFORMATION: Clone 3 forward primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 114: CCTGACCTGA TCAAGTTTA 19 (2)INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...18 (D) OTHER INFORMATION: Clone 3 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: GGTAAAGGAA CAAGCTGC 18(2) INFORMATION FOR SEQ ID NO: 116: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 4 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: TGATCACACA GTCAGCACTGT 21 (2) INFORMATION FOR SEQ ID NO: 117: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 4 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: GGGCAGAAGT TTCCAATTACC 21 (2) INFORMATION FOR SEQ ID NO: 118: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...23 (D) OTHER INFORMATION: Clone 5 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: TATTGAGACC TAAGTCAGCATCC 23 (2) INFORMATION FOR SEQ ID NO: 119: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 5 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: GACAGAAAGC AGGTTAGAGGT 21 (2) INFORMATION FOR SEQ ID NO: 120: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: Clone 6 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: GAAACTTTAC ATCAGGTGTCTC 22 (2) INFORMATION FOR SEQ ID NO: 121: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 6 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: ATGGACTAGG AGTTTAAGC19 (2) INFORMATION FOR SEQ ID NO: 122: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 7 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122: GGAACAGTGT ACACTTTCC19 (2) INFORMATION FOR SEQ ID NO: 123: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: Clone 7 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123: TATATAGCCT CGATGATGAGAG 22 (2) INFORMATION FOR SEQ ID NO: 124: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: Clone 8 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124: CATGAGAGGA AGAGGTCTTTAT 22 (2) INFORMATION FOR SEQ ID NO: 125: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 8 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: GGGTTATGTC TTAGTCGAG19 (2) INFORMATION FOR SEQ ID NO: 126: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...23 (D) OTHER INFORMATION: Clone 9 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: TCAGTAGAAA CTCAAGCTGCTTC 23 (2) INFORMATION FOR SEQ ID NO: 127: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 9 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: CTCCCTCTCA GTGTGAGGCT20 (2) INFORMATION FOR SEQ ID NO: 128: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 10 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128: CCTGACCTGA TCAAGTTTAA20 (2) INFORMATION FOR SEQ ID NO: 129: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 10 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: TGTACACCAC TCCTCATGT19 (2) INFORMATION FOR SEQ ID NO: 130: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 11 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: CGACGACTCA TACAACATATC 21 (2) INFORMATION FOR SEQ ID NO: 131: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 11 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: GGTTACAGCT GAAGTGTAT19 (2) INFORMATION FOR SEQ ID NO: Clone 12 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 132: TATTCAGGAA CAGTGTACAC 20 (2) INFORMATION FOR SEQ ID NO:Clone 12 reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133: TCGATGATGAGAGGGTTAC 19 (2) INFORMATION FOR SEQ ID NO: 134: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION:Clone 13 forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:GAACACTTAT CTCCTTCTTC AG 22 (2) INFORMATION FOR SEQ ID NO: 135: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (D) OTHERINFORMATION: Clone 13 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 135: TCCACTCCTT TCACCTCTTC T 21 (2) INFORMATION FOR SEQ ID NO: 136:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHERINFORMATION: Clone 14 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 136: AGACAAGAGC AAAACACAAC 20 (2) INFORMATION FOR SEQ ID NO: 137:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: Clone 14 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 137: CTCTTTGCAG TTCAGTCTA 19 (2) INFORMATION FOR SEQ ID NO: 138: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: Clone 15 forward primer (ix) FEATURE: (A) NAME/KEY:modified_base (B) LOCATION: 4 (D) OTHER INFORMATION: N = inosine (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 138: AGGNGAACCA TTTGACTGGT TT 22 (2)INFORMATION FOR SEQ ID NO: 139: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 15 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: GCTTGTGTGT GGCTGTCCTT20 (2) INFORMATION FOR SEQ ID NO: 140: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...25 (D) OTHER INFORMATION: Clone 16 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: GGCTAAACTT ACAGTATGTAAGGAG 25 (2) INFORMATION FOR SEQ ID NO: 141: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION:Clone 16 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:CTGTAAGGAC AGACTACTCA 20 (2) INFORMATION FOR SEQ ID NO: 142: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...17 (D) OTHERINFORMATION: Clone 17 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 142: CCAGGAGGTT CAGCGGT 17 (2) INFORMATION FOR SEQ ID NO: 143: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHERINFORMATION: Clone 17 reverse primer (ix) FEATURE: (A) NAME/KEY:Modified Base (B) LOCATION: 14 (D) OTHER INFORMATION: N = inosine (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 143: CGCAAAGCCA TGANAAACCG 20 (2)INFORMATION FOR SEQ ID NO: 144: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 18 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: TCAGGAACAG TGTACACTTTC 21 (2) INFORMATION FOR SEQ ID NO: 145: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 18 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: TGTGGGCTTA ATACCATGTCT 21 (2) INFORMATION FOR SEQ ID NO: 146: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...18 (D) OTHER INFORMATION: Clone 19 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: GGAATCTCTG TACTTGCT 18(2) INFORMATION FOR SEQ ID NO: 147: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 19 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: GTGACACATT ACAAAGCCA19 (2) INFORMATION FOR SEQ ID NO: Clone 20 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 148: TCAGTAGAAA CTCAAGCTGC 20 (2) INFORMATION FOR SEQ ID NO:Clone 20 reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: CCTCTTCCTCTTAAAGTGT 19 (2) INFORMATION FOR SEQ ID NO: Clone 21 forward primer (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 150: TCACTTCAGA ATCACTACTC 20 (2) INFORMATIONFOR SEQ ID NO: Clone 21 reverse primer (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151: ACCCATCCTA TATGAAAAGC 20 (2) INFORMATION FOR SEQ ID NO: Clone 23forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152: GGGATCATAC TAAAGAGAAG20 (2) INFORMATION FOR SEQ ID NO: Clone 23 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 153: GGATAAACAG AGAGCTTGAT 20 (2) INFORMATION FOR SEQ ID NO:Clone 25 forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: GTCAGTTACTCTATTTGCTG TG 22 (2) INFORMATION FOR SEQ ID NO: Clone 25 reverse primer(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 155: AACCTGTGCT GTAAAGTTCA 20 (2)INFORMATION FOR SEQ ID NO: Clone 26 forward primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 156: CTTAAGAGGA AGAGGCCAT 19 (2) INFORMATION FOR SEQ ID NO:Clone 26 reverse primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157: CTCTCCCTCTCAGTGTGAG 19 (2) INFORMATION FOR SEQ ID NO: Clone 27 forward primer (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 158: ACAATTAGGC ATTGTTGATG G 21 (2) INFORMATIONFOR SEQ ID NO: Clone 27 reverse primer (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159: CAGNTCTTGC ACATACAAGA CA 22 (2) INFORMATION FOR SEQ ID NO: Clone 28forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: ACCTTTGGCA AGGGGTATGA20 (2) INFORMATION FOR SEQ ID NO: Clone 28 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 161: TGTGAAGGCT GGGAAACACT 20 (2) INFORMATION FOR SEQ ID NO:162: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: Clone 30 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 162: AACACTCAGC TCTGTAGAA 19 (2) INFORMATION FOR SEQ ID NO: 163: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: Clone 30 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 163: CGAGTCATCA ATAGGACAA 19 (2) INFORMATION FOR SEQ ID NO: Clone 32forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:1...21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164: GAGCCAAGTG GAACTCTTGAA 21 (2) INFORMATION FOR SEQ ID NO: Clone 32 reverse primer (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 165: GTCAGGAAAG AGGTTGTGAG C 21 (2) INFORMATION FOR SEQ IDNO: 166: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20(D) OTHER INFORMATION: Clone 33 forward primer (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 166: ACACATATGT ACACAGGAAC 20 (2) INFORMATIONFOR SEQ ID NO: 167: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...20 (D) OTHER INFORMATION: Clone 33 reverse primer (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 167: TGTGTACAGC GAGTGAATTA 20 (2)INFORMATION FOR SEQ ID NO: 168: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 34 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168: TTGTTCACAC ACAATCTAGG20 (2) INFORMATION FOR SEQ ID NO: 169: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 34 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169: ACTAGCATAT CTGAATTCCCA 21 (2) INFORMATION FOR SEQ ID NO: 170: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: Clone 35 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: CTACAGAATA GAATACATGGCG 22 (2) INFORMATION FOR SEQ ID NO: 171: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 35 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171: TTGAAACCAG ACCCTGTAGT20 (2) INFORMATION FOR SEQ ID NO: 172: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 36 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172: CATTTAGTCC AGAGGCTCTT20 (2) INFORMATION FOR SEQ ID NO: 173: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 36 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173: TCCTCGAAGA GGTTGCAGC19 (2) INFORMATION FOR SEQ ID NO: 174: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION: Clone 38 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174: CATTCAGCAC ACATAGAGTCTA 22 (2) INFORMATION FOR SEQ ID NO: 175: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 38 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175: CCCTGTCCCT TGTATATGTA20 (2) INFORMATION FOR SEQ ID NO: 176: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...24 (D) OTHER INFORMATION: Clone 39 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176: AGTGTATCTA CAACCTCAACTGTC 24 (2) INFORMATION FOR SEQ ID NO: 177: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHER INFORMATION:Clone 39 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177:GTAAAGGCCC AATCAATGCA CT 22 (2) INFORMATION FOR SEQ ID NO: 178: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (D) OTHERINFORMATION: Clone 40 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 178: GCCAGATTCA CAATTGATAG 20 (2) INFORMATION FOR SEQ ID NO: 179:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...19 (D) OTHERINFORMATION: Clone 40 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 179: CTGAAGGCAC TTTATGTAC 19 (2) INFORMATION FOR SEQ ID NO: 180: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...21 (D) OTHERINFORMATION: Clone 41 forward primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 180: CTGGAGCAGG TTAGATACAC C 21 (2) INFORMATION FOR SEQ ID NO: 181:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...22 (D) OTHERINFORMATION: Clone 41 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 181: CTTCCCTCTT AACCTTTAGT GC 22 (2) INFORMATION FOR SEQ ID NO:Clone 42 forward primer (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B)LOCATION: 1...21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182: GTGTCTTGTATGTGCAAGAA C 21 (2) INFORMATION FOR SEQ ID NO: Clone 42 reverse primer(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...20 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 183: GACTGGGTAT CCTAGCTTAC 20 (2)INFORMATION FOR SEQ ID NO: 184: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 43 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184: TTAGTCAGAC CCATTCAGTC20 (2) INFORMATION FOR SEQ ID NO: 185: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 43 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 185: CCAGACTGCT TTATGTTAG19 (2) INFORMATION FOR SEQ ID NO: 186: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...21 (D) OTHER INFORMATION: Clone 44 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 186: GTGTCTTGTA TGTGCAAGAAC 21 (2) INFORMATION FOR SEQ ID NO: 187: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 44 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187: CCTAGCCTTA CTGTTTTAAC20 (2) INFORMATION FOR SEQ ID NO: 188: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...18 (D) OTHER INFORMATION: Clone 45 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 188: ACGATGCGAT CCTGGAAG 18(2) INFORMATION FOR SEQ ID NO: 189: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...18 (D) OTHER INFORMATION: Clone 45 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189: CTGGCTTGAG TTTGTCTG 18(2) INFORMATION FOR SEQ ID NO: 190: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 46 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 190: CCTTTCTGTG TGAAGATCAC20 (2) INFORMATION FOR SEQ ID NO: 191: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 46 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191: AAGAAAGTCC CAAGGGTGGA20 (2) INFORMATION FOR SEQ ID NO: 192: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...19 (D) OTHER INFORMATION: Clone 47 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 192: GGAATGAGGG TTAGAGTCC19 (2) INFORMATION FOR SEQ ID NO: 193: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...18 (D) OTHER INFORMATION: Clone 47 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 193: AGTGCTTCTG TAGCTCTT 18(2) INFORMATION FOR SEQ ID NO: 194: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 48 forwardprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 194: TGAGGGTGTG AACCACTCTG20 (2) INFORMATION FOR SEQ ID NO: 195: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A)NAME/KEY: - (B) LOCATION: 1...20 (D) OTHER INFORMATION: Clone 48 reverseprimer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 195: GAATCCTGGT GTGCCCAAGT20 (2) INFORMATION FOR SEQ ID NO: 196: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 13...14 (D) OTHER INFORMATION: Xaa= Gly or Ala (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 196: Trp Asp Xaa XaaAla Ala Xaa Val Ile Xaa Xaa Xaa Xaa Xaa 1 5 10 (2) INFORMATION FOR SEQID NO: 197: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1...17(D) OTHER INFORMATION: primer used for primer extension analysis (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 197: GGGCGACCGA CGGGAAG 17

What is claimed is:
 1. A method for determining a genotype associatedwith increased susceptibility to manic-depressive illness, comprisingdetermining the genotype of an affected individual with at least onepolymorphic marker localized within the chromosomal region defined byand including markers D18S843 and D18S869 and determining therefrom thegenotype associated with increased susceptibility to manic-depressivedisorder.
 2. The method of claim 1, wherein said polymorphic marker isamplified by primers which selectively hybridize, under stringentconditions, to the same nucleic acid sequences as primers of SEQ ID NO:1and SEQ ID NO:2.
 3. The method of claim 1, wherein said polymorphicmarker is amplified by the polymerase chain reaction.
 4. The method ofclaim 1, further comprising: determining the genotype of a testedindividual wherein the genotype is determined with at least onepolymorphic marker localized within the chromosomal region defined byand including markers D18S843 and D18S869; comparing the genotype of thetested individual to the genotype associated with increasedsusceptibility to manic-depressive illness; and determining therefromthe increased or decreased risk of the tested individual developingmanic-depressive illness.
 5. The method of claim 4, wherein saidpolymorphic marker is amplified by primers which selectively hybridize,under stringent conditions, to the same nucleic acid sequences asprimers of SEQ ID NO:1 and SEQ ID NO:2.
 6. A nucleic acid composition,comprising oligonucleotide primers which selectively hybridize, understringent conditions, to the same nucleic acid sequence as primers ofSEQ ID NO:1 and SEQ ID NO:2.
 7. An isolated nucleic acid of less than 10kB in length and comprising a polymorphic marker amplified byoligonucleotide primers of SEQ ID NO:1 and SEQ ID NO:2.
 8. A method fordetermining an increased susceptibility to manic-depressive illness in atested individual, comprising determining the genotype of the testedindividual with oligonucleotide primers which amplify the samepolymorphic marker as primers of SEQ ID NO:1 and SEQ ID NO:2, whereinthe presence of allele 2 of the polymorphic marker indicates anincreased susceptibility to manic-depressive illness.
 9. An isolatednucleic acid encoding an IMP.18p myo-inositol monophosphatase, saidprotein defined as follows: (i) having a calculated molecular weight ofbetween about 22 to 34 kDa; (ii) the protein's activity includeshydrolysis of myo-inositol 1-phosphate to generate inositol andinorganic phosphate; and (iii) (a) specifically binding to an antibodyraised against an IMP.18p myo-inositol monophosphatase protein, orimmunogenic fragment thereof, consisting of SEQ ID NO:17; or (b) havingat least 60% amino acid sequence identity to an IMP.18p myo-inositolmonophosphatase protein consisting of SEQ ID NO:17, as measured using asequence comparison algorithm.
 10. The isolated nucleic acid of claim 9,wherein the calculated molecular weight of the encoded protein is about28 to 29 kDa.
 11. The isolated nucleic acid of claim 9, wherein theencoded protein has at least 80% amino acid sequence identity to theIMP.18p myo-inositol monophosphatase protein of SEQ ID NO. 17, asmeasured using a sequence comparison algorithm.
 12. The isolated nucleicacid of claim 9, wherein the encoded protein has the sequence set forthin SEQ ID NO:17.
 13. An isolated nucleic acid which specificallyhybridizes to SEQ ID NO:16 under stringent conditions.
 14. An isolatednucleic acid encoding an IMP.18p myo-inositol monophosphatase proteinwhich specifically binds to an antibody directed against a proteinhaving a sequence as set forth in SEQ ID NO:17.
 15. A polynucleotide orfragment thereof comprising a purified antisense nucleotide capable ofhybridizing to and having a nucleic acid sequence complementary to atleast a portion of an IMP.18p myo-inositol monophosphatasepolynucleotide encoding an IMP.18p myo-inositol monophosphatase protein,said protein defined as follows: (i) having a calculated molecularweight of between about 22 to 34 kDa; (ii) the protein's activityincludes hydrolysis of myo-inositol 1-phosphate to generate inositol andinorganic phosphate; and (iii) (a) specifically binding to an antibodyraised against an IMP.18p myo-inositol monophosphatase protein, orimmunogenic fragment thereof, consisting of SEQ ID NO:17; or (b) havingat least 60% amino acid sequence identity to an IMP.18p myo-inositolmonophosphatase protein consisting of SEQ ID NO:17, as measured using asequence comparison algorithm.
 16. An expression vector comprising anucleic acid encoding: an IMP.18p myo-inositol monophosphatase protein,said protein defined as follows (i) having a calculated molecular weightof between about 22 to 34 kDa, (ii) the protein's activity includeshydrolysis of myo-inositol l-phosphate to generate inositol andinorganic phosphate, and (iii) (a) specifically binding to an antibodyraised against an IMP.18p myo-inositol monophosphatase protein, orimmunogenic fragment thereof, consisting of SEQ ID NO:17, or (b) havingat least 60% amino acid sequence identity to an IMP.18p myo-inositolmonophosphatase protein consisting of SEQ ID NO:17, as measured using asequence comparison algorithm; or, an antisense nucleic acid sequencecomplementary to at least a portion of the IMP.18p myo-inositolmonophosphatase polynucleotide.
 17. A cell comprising an exogenousnucleic acid sequence comprising a nucleic acid which encodes: anIMP.18p myo-inositol monophosphatase protein, said protein defined asfollows (i) having a calculated molecular weight of between about 22 to34 kDa, (ii) the protein s activity includes hydrolysis of myo-inositol1-phosphate to generate inositol and inorganic phosphate, and (iii) (a)specifically binding to an antibody raised against an IMP.18pmyo-inositol monophosphatase protein, or immunogenic fragment thereof,consisting of SEQ ID NO:17, or (b) having at least 60% amino acidsequence identity to an IMP.18p myo-inositol monophosphatase proteinconsisting of SEQ ID NO:17, as measured using a sequence comparisonalgorithm; or, an antisense nucleic acid sequence complementary to atleast a portion of the IMP.18p myo-inositol monophosphatasepolynucleotide.
 18. An organism comprising an exogenous nucleic acidsequence, wherein the nucleic acid specifically hybridizes understringent conditions to SEQ ID NO:16 or comprises the nucleic acidencoding an IMP.18p myo-inositol monophosphatase protein, said proteindefined as follows (i) having a calculated molecular weight of betweenabout 22 to 34 kDa, (ii) the protein's activity includes hydrolysis ofmyo-inositol 1-phosphate to generate inositol and inorganic phosphate,and (iii) (a) specifically binding to an antibody raised against anIMP.18p myo-inositol monophosphatase protein, or immunogenic fragmentthereof, consisting of SEQ ID NO:17, or (b) having at least 60% aminoacid sequence identity to an IMP.18p myo-inositol monophosphataseprotein consisting of SEQ ID NO:17, as measured using a sequencecomparison algorithm; or fragment thereof, has been introduced, and theorganism expresses the exogenous nucleic acid as an IMP.18p myo-inositolmonophosphatase protein, or fragment thereof.
 19. The organism of claim17, wherein the exogenous nucleic acid sequence is translated into anIMP.18p myo-inositol monophosphatase protein which is expressedexternally from the organism.
 20. An isolated IMP.18p myo-inositolmonophosphatase protein, said protein: (i) having a calculated molecularweight of about 22 to 34 kDa; (ii) the protein's activity includeshydrolysis of myo-inositol 1-phosphate to generate inositol andinorganic phosphate; and (v) (a) specifically binding to an antibodyraised against a myo-inositol monophosphatase protein, or immunogenicfragment thereof, consisting of SEQ ID NO:17; or (b) having at least 60%amino acid sequence identity to a myo-inositol monophosphatase proteinconsisting of SEQ ID NO:17, as measured using a sequence comparisonalgorithm.
 21. The isolated IMP.18p myo-inositol monophosphatase proteinof claim 20, wherein the myo-inositol monophosphatase protein can befound in humans.
 22. The isolated IMP.18p myo-inositol monophosphataseprotein of claim 20, wherein the calculated molecular weight is about 28to 29 kDa.
 23. The isolated IMP.18p myo-inositol monophosphatase proteinof claim 20, wherein the protein has a sequence as set forth in SEQ IDNO:17.
 24. An isolated antibody, specifically immunoreactive underimmunologically reactive conditions, to an IMP.18p myo-inositolmonophosphatase protein, said protein having the sequence as set forthin SEQ ID NO:17.
 25. An isolated antibody, specifically immunoreactiveunder immunologically reactive conditions, to a protein defined asfollows (i) having a calculated molecular weight of between about 22 to34 kDa, (ii) the protein's activity includes hydrolysis of myo-inositoll-phosphate to generate inositol and inorganic phosphate, and (iii) (a)specifically binding to an antibody raised against an IMP.18pmyo-inositol monophosphatase protein, or immunogenic fragment thereof,consisting of SEQ ID NO:17, or (b) having at least 60% amino acidsequence identity to an IMP.18p myo-inositol monophosphatase proteinconsisting of SEQ ID NO:17, as measured using a sequence comparisonalgorithm.
 26. A pharmaceutical composition comprising an acceptablecarrier and an IMP.18p myo-inositol monophosphatase protein as definedby the protein of claim 20; an anti-IMP.18p myo-inositol monophosphataseantibody or binding fragment thereof as defined by claims 24 and 25; ora polynucleotide encoding an IMP.18p myo-inositol monophosphataseprotein as defined by claim
 9. 27. A method for quantifying the amountof a myo-inositol monophosphatase in a mammal, comprising: (a) obtaininga cell or tissue sample from the mammal; and (b) determining the amountof an IMP.18p myo-inositol monophosphatase gene product in the cell ortissue.
 28. A method for detecting the presence of a polynucleotidesequence encoding at least a portion of an IMP.18p myo-inositolmonophosphatase in a biological sample, comprising the steps of: a)providing: i) a biological sample suspected of containing a nucleic acidcorresponding to the polynucleotide sequence of an IMP.18p myo-inositolmonophosphatase; ii) a probe comprising a nucleotide sequence of anIMP.18p myo-inositol monophosphatase, or a fragment thereof capable ofhybridizing to a myo-inositol monophosphatase-encoding nucleic acid,from a biological sample; b) combining said nucleic acid-containingbiological sample with said probe under conditions such that ahybridization complex is formed between said nucleic acid and saidprobe; and c) detecting said hybridization complex.
 29. The method ofclaim 28, wherein, said nucleic acid in said biological sample isribonucleic acid.
 30. The method of claim 29, wherein said detectedhybridization complex correlates with expression of an IMP.18pmyo-inositol monophosphatase in said biological sample.
 31. A method ofdetermining whether a test compound is a modulator of an IMP.18pmyo-inositol monophosphatase activity, said method comprising the stepsof: a) providing a composition comprising an IMP.18p myo-inositolmonophosphatase protein; b) contacting the IMP.18p myo-inositolmonophosphatase protein with the test compound, and c) measuring theactivity of the IMP.18p myo-inositol monophosphatase, wherein a changein the IMP.18p myo-inositol monophosphatase activity in the presence ofthe test compound is an indicator of whether the test compound modulatesthe IMP.18p myo-inositol monophosphatase activity.
 32. The method ofclaim 31, wherein the IMP.18p myo-inositol monophosphatase protein isdefined as follows (i) having a calculated molecular weight of betweenabout 22 to 34 kDa, (ii) the protein's activity includes hydrolysis ofmyo-inositol 1-phosphate to generate inositol and inorganic phosphate,and (iii) (a) specifically binding to an antibody raised against anIMP.18p myo-inositol monophosphatase protein, or immunogenic fragmentthereof, consisting of SEQ ID NO:17, or (b) having at least 60% aminoacid sequence identity to an IMP.18p myo-inositol monophosphataseprotein consisting of SEQ ID NO:17, as measured using a sequencecomparison algorithm.
 33. The method of claim 31, wherein thecomposition comprises a cell.
 34. The method of claim 31, wherein thecomposition comprises an organism.